Conantokins

ABSTRACT

The present invention is directed to conantokin peptides, conantokin peptide derivatives and conantokin peptide chimeras, referred to collectively as conantokins, having 10-30 amino acids, including preferably two or more gamma-carboxyglutamic acid residues. The conantokins are useful for the treatment of neurologic and psychiatric disorders, such as anticonvulsant agents, neuroprotective agents or analgesic agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 35 U.S.C. §371 of PCT/US/97/12618 filed onJul. 21, 1997, which is a continuation-in-part application of U.S.patent application Ser. No. 08/684,742 filed on Jul. 22, 1996 nowabandoned.

This invention was made with Government support under Grant No. PO1GM48677 awarded by the National Institute of General Medical Sciences,National Institutes of Health, Bethesda, Md. The United StatesGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

The invention relates to relatively short peptides (termed conantokinsherein), about 10-30 residues in length, which are naturally availablein minute amounts in the venom of the cone snails or analogous to thenaturally available-peptides, and which include preferably one to two ormore γ-carboxyglutamic acid residues. The conantokins are useful for thetreatment of neurologic and psychiatric disorders, such asanticonvulsant agents, as neuroprotective agents or for the managementof pain.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference, and for convenience are referenced in the following text byauthor and date and are listed alphabetically by author in the appendedbibliography.

The predatory cone snails (Conus) have developed a unique biologicalstrategy. Their venom contains relatively small peptides that aretargeted to various neuromuscular receptors and may be equivalent intheir pharmacological diversity to the alkaloids of plants or secondarymetabolites of microorganisms. Many of these peptides are among thesmallest nucleic acid-encoded translation products having definedconformations, and as such, they are somewhat unusual. Peptides in thissize range normally equilibrate among many conformations. Proteinshaving a fixed conformation are generally much larger.

The cone snails that produce these peptides are a large genus ofvenomous gastropods comprising approximately 500 species. All cone snailspecies are predators that inject venom to capture prey, and thespectrum of animals that the genus as a whole can envenomate is broad. Awide variety of hunting strategies are used, however, every Conusspecies uses fundamentally the same basic pattern of envenomation.

Several peptides isolated from Conus venoms have been characterized.These include the α-, μ- and ω-conotoxins which target nicotinicacetylcholine receptors, muscle sodium channels, and neuronal calciumchannels, respectively (Olivera et al., 1985). Conopressins, which arevasopressin analogs, have also been identified (Cruz et al., 1987). Inaddition, peptides named conantokins have been isolated from Conusgeographuis and Conus tulipa (Mena et al., 1990; Haack et al., 1990).These peptides have unusual age-dependent physiological effects: theyinduce a sleep-like state in mice younger than two weeks and hyperactivebehavior in mice older than 3 weeks (Haack et al., 1990).

The conantokins are structurally unique. In contrast to the wellcharacterized conotoxins from Conus venoms, most conantokins do notcontain disulfide bonds. However, they contain 4-5 residues of theunusual modified amino acid γ-carboxyglutamic acid. The occurrence ofthis modified amino acid, which is derived post-translationally fromglutamate in a vitamin K-dependent reaction, was unprecedented in aneuropeptide. It has been established that the conantokins haveN-methyl-D-aspartate (NMDA) antagonist activity, and consequently targetthe NMDA receptor. The conantokins reduce glutamate (or NMDA) mediatedincreases in intracellular Ca²⁺ and cGMP without affectingkainate-mediated events (Chandler et al., 1993). Although these peptideshave actions through polyamine responses of the NMDA receptors, theneurochemical profile of these polypeptides is distinct from previouslydescribed noncompetitive NMDA antagonists (Skolnick et al., 1992).

The previously identified conantokins are Conantokin G (Con G) andConantokin T (Con T). Con G has the formulaGly-Glu-Xaa₁-Xaa₁-Leu-Gln-Xaa₂-Asn-Gln-Xaa₂-Leu-Ile-Arg-Xaa₂-Lys-Ser-Asn(SEQ ID NO:1), wherein Xaa₁ and Xaa₂ are γ-carboxyglutamic acid (Gla).The C-terminus preferably contains an amide group. Con T has the formulaGly-Glu-Xaa₁-Xaa₁-Tyr-Gln-Lys-Met-Leu-Xaa₂-Asn-Leu-Arg-Xaa₂-Ala-Glu-Val-Lys-Lys-Asn-Ala(SEQ ID NO:2), wherein Xaa₁ and Xaa₂ are γ-carboxyglutamic acid. TheC-terminus preferably contains an amide group. Analogues of Conantokin Ghave been synthesized and analyzed for their biological activity(Chandler et al., 1993; Zhou et al., 1996). It has been discovered thatsubstitution of the Gla residue at position 4 of Con G destroys its NMDAantagonist properties. Substitution of the Gla residue at position 3 ofCon G greatly reduces its NMDA antagonist activity. However,substitutions of the Gla residues at positions 7, 10 and 14 of Con G donot adversely affect potency of the peptide and may even enhance it.(Zhou et al., 1996).

Ischemic damage to the central nervous system (CNS) may result formeither global or focal ischemic conditions. Global ischemia occurs underconditions in which blood flow to the entire brain ceases for a periodof time, such as may result from cardiac arrest. Focal ischemia occursunder conditions in which a portion of the brain is deprived of itsnormal blood supply, such as may result from thromboembolytic occlusionof a cerebral vessel, traumatic head or spinal cord injury, edema orbrain or spinal cord tumors. Both global and focal ischemic conditionshave the potential for widespread neuronal damage, even if the globalischemic condition is transient or the focal condition affects a verylimited area.

Epilepsy is a recurrent paroxysmal disorder of cerebral functioncharacterized by sudden brief attacks of altered consciousness, motoractivity, sensory phenomena or inappropriate behavior caused by abnormalexcessive discharge of cerebral neurons. Convulsive seizures, the mostcommon form of attacks, begin with loss of consciousness and motorcontrol, and tonic or clonic jerking of all extremities but anyrecurrent seizure pattern may be termed epilepsy. The term primary oridiopathic epilepsy denotes those cases where no cause for the seizurescan be identified. Secondary or symptomatic epilepsy designates thedisorder when it is associated with such factors as trauma, neoplasm,infection, developmental abnormalities, cerebrovascular disease, orvarious metabolic conditions. Epileptic seizures are classified aspartial seizures (focal, local seizures) or generalized seizures(convulsive or nonconvulsive). Classes of partial seizures includesimple partial seizures, complex partial seizures and partial seizuressecondarily generalized. Classes of generalized seizures include absenceseizures, atypical absence seizures, myoclonic seizures, clonicseizures, tonic seizures, tonic-clonic seizures (grand mal) and atonicseizures. Therapeutics having anticonvulsant properties are used in thetreatment of seizures. Most therapeutics used to abolish or attenuateseizures act at least through effects that reduce the spread ofexcitation from seizure foci and prevent detonation and disruption offunction of normal aggregates of neurons. Traditional anticonvulsantsthat have been utilized include phenytoin, phenobarbital, primidone,carbamazepine, ethosuximide, clonazepam and valproate. Several novel andchemically diverse anticonvulsant medications recently have beenapproved for marketing, including lamotrigine, feribamate, gabapentinand topiramate. For further details of seizures and their therapy, seeRall & Schleifer (1985) and The Merck Manual (1992).

It has been shown that neurotransmission mediated through the NMDAreceptor complex is associated with seizures (Bowyer, 1982; McNamara etal., 1988), ischemic neuronal injury (Simon et al., 1984; Park et al.,1988) and other phenomena including synaptogenesis (Cline et al., 1987),spatial learning (Morris et al., 1986) and long-term potentiation(Collinridge et al., 1983; Harris et al., 1984; Morris et al., 1986).Regulation of these neuronal mechanisms by NMDA-mediated processes mayinvolve activation of a receptor-gated calcium ion channel (Nowak etal., 1984; Mayer et al., 1987; Ascher and Nowak, 1988).

The NMDA channel is regulated by glycine. This amino acid increasesNMDA-evoked currents in various tissues [Johnson and Ascher, 1987;Kleckner and Dingledine, 1988] by increasing the opening frequency ofthe NMDA channel [Johnson and Ascher, 1987]. Thus, NMDA-induced calciuminflux and intracellular accumulation may be stimulated by glycine[Reynolds et al., 1987; Wroblewski et al., 1989], which interacts withits own distinct site [Williams et al., 1991]. Furthermore, accumulationof intracellular calcium may be implicated in the aforementionedneuropathologies.

The NMDA receptor is also involved in a broad spectrum of CNS disorders.For example, during brain ischemia caused by stroke or traumatic injury,excessive amounts of the excitatory amino acid glutamate are releasedfrom damaged or oxygen deprived neurons. This excess glutamate binds theNMDA receptor which opens the ligand-gated ion channel thereby allowingCa²⁺ influx producing a high level of intracellular Ca²⁺, whichactivates biochemical cascades resulting in protein, DNA and membranedegradation leading to cell death. This phenomenon, known asexcitotoxicity, is also thought to be responsible for the neurologicaldamage associated with other disorders ranging from hypoglycemia andcardiac arrest to epilepsy. In addition, there are reports indicatingsimilar involvement in the chronic neurodegeneration of Huntington's,Parkinson's and Alzheimer's diseases.

Parkinson's disease is a progressive, neurodegenerative disorder. Theetiology of the disorder is unknown in most cases, but has beenhypothesized to involve oxidative stress. The underlying neuropathologyin Parkinsonian patients is an extensive degenerations of the pigmenteddopamine neurons in the substantia nigra. These neurons normallyinnervate the caudate and putamen nuclei. Their degeneration results ina marked loss of the neurotransmitter dopamine in the caudate andputamen nuclei. This loss of dopamine and its regulation of neurons inthe caudate-putamen leads to the bradykinesia, rigidity, and tremor thatare the hallmarks of Parkinson's disease. An animal model has beendeveloped for Parkinson's disease (Zigmond et al., 1987) and has beenused to test agents for anti-Parkinsonian activity (Ungerstedt et al.,1973).

The dopamine precursor, L-Dopa, is the current therapy of choice intreating the symptoms of Parkinson's disease. However, significant sideeffects develop with continued use of this drug and with diseaseprogression, making the development of novel therapies important.Recently, antagonists of the NMDA subtype of glutamate receptor havebeen proposed as potential anti-Parkinsonian agents. (Borman, 1989;Greenamyre and O'Brien, 1991; Olney et al., 1987). In addition,antagonists of NMDA receptors potentiate the behavioral effects ofL-Dopa and D1 dopamine receptor stimulation in animal models ofParkinson's disease. (Starr, 1995). These data suggest that NMDAreceptor antagonists may be useful adjuncts to L-Dopa therapy inParkinson's disease by decreasing the amount of L-Dopa required andthereby reducing undesirable side effects. In addition, antagonists ofNMDA receptors have been shown to attenuate free radical mediatedneuronal death. Thus, NMDA receptor antagonists may also prevent furtherdegeneration of dopamine neurons in addition to providing symptomaticrelief. Finally, NMDA receptor antagonists have been shown to potentiatethe contralateral rotations induced by L-Dopa or D1 dopamine receptorantagonists in the animal model.

Pain, and particularly, persistent pain, is a complex phenomenoninvolving many interacting components. Numerous studies, however, havedemonstrated a role for NMDA receptors in mediating persistent pain, andfurther that NMDA antagonists are effective in animal models ofpersistent pain. First, administration of NMDA (the agonist) mimics manyof the physiological and behavioral effects of painful stimuli (Chapmanet al., 1994; Dougherty and Willis, 1991; Coderre and Melzack, 1992;Malmberg and Yaksh, 1992; Bach et al., 1994; Liu et al., 1997). Second,various classes of NMDA antagonists block the “wind up” (progressiveaugmentation of response caused by repetitive stimulation) of smallprimary afferent C fibers of the dorsal horn (Davies and Lodge, 1987;Dickenson and Sullivan, 1987; Thompson et al., 1990). Third, release ofglutamate and aspartate (agonists at NMDA and non-NMDA glutamatergicreceptors) is increased in spinal cord in animal models of persistentpain (Sluka and Westlund, 1992; Malmberg and Yaksh, 1992; Yang et al.,1995). Fourth, NMDA antagonists are effective in animal models ofpersistent pain (Neugebauer et al., 1993; Coderre, 1993; Coderre and VanEmpel, 1994; Yamamoto and Yaksh, 1992; Chaplan et al., 1997; Millan andSeguin, 1994; Rice and McMahon, 1994). Moreover, NMDA antagonists appearto be effective in reducing the tolerance to opioid analgesics seenafter chronic administration in animal models of pain (Bilsky et al.,1996; Lufty et al., 1996; Shimoyama et al., 1996; Wong et al., 1996;Elliot et al., 1994; Mao et al., 1994; Dunbar and Yaksh, 1996; Lufty etal., 1995; Trujillo and Akil, 1994; Tiseo et al., 1994; Gutstein andTrujillo, 1993; Kest et al., 1993; Tiseo and Inturrisi, 1993). Finally,severe or prolonged tissue or nerve injury can induce hyperexcitabilityof dorsal horn neurons of the spinal cord, resulting in persistent pain,an exacerbated response to noxious stimuli (hyperalgesia) and a loweredpain threshold (allodynia). These changes are mediated by NMDA-typeglutamate receptors in the spinal cord, whose activation causes releaseof Substance P, a peptide neurotransmitter made by small-diameter,primary, sensory pain fibres. Injection of NMDA in the cerebrospinalfluid of the rat spinal cord mimicked the changes that occur withpersistent injury and produced pain (Liu et al., 1997).

Neuropsychiatric involvement of the NMDA receptor has also beenrecognized. Blockage of the NMDA receptor Ca2+ channel by the animalanesthetic phencyclidine produces a psychotic state in humans similar toschizophrenia (Johnson et al., 1990). Further, NMDA receptors have alsobeen implicated in certain types of spatial learning (Bliss et al.,1993). In addition, numerous studies have demonstrated a role for NMDAreceptors in phenomena associated with addiction to and compulsive useof drugs or ethanol. Furthermore, antagonists of NMDA receptors may beuseful for treating addiction-related phenomena such as tolerance,sensitization, physical dependence and craving (for review see, Popik etal., 1995; Spanagel and Zieglgansberger, 1997; Trujillo and Akil, 1995).

There are several lines of evidence which suggest that NMDA antagonistsmay be useful in the treatment of HIV infection. First, the levels ofthe neurotoxin and NMDA agonist quinolinic acid are elevated in thecerebrospinal fluid of HIV-positive subjects (Heyes et al., 1989) and inmurine retrovirus-induced immunodeficiency syndrome (Sei et al., 1996).Second, the envelope glycoprotein of HIV-1 alters NMDA receptor function(Sweetnam et al., 1993). Thirdly, NMDA antagonists can reduce theeffects and neurotoxicity of GP-120 (Muller et al., 1996; Raber et al.,1996; Nishida et al., 1996). Fourth, GP-120 and glutamate actsynergistically to produce toxicity in vitro (Lipton et al., 1991). Andfinally, memantine, an NMDA antagonist, protects against HIV infectionin glial cells in vitro (Rytik et al., 1991). For a review of the use ofNMDA antagonists in treating HIV infection, see Lipton (1994; 1996).

It is desired to identify additional conantokin peptides and relatedcompounds which target the NMDA receptor. It is further desired toidentify compounds which are useful as anticonvulsant, neuroprotective,neuropsychiatric or analgesic agents.

SUMMARY OF THE INVENTION

The present invention is directed to conantokin peptides, conantokinpeptide derivatives and conantokin peptide chimeras, referred tocollectively as conantokins (unless the context dictates otherwise),having 10-30 amino acids, including preferably one to two or moreγ-carboxyglutamic acid residues. The conantokins are useful for thetreatment of neurologic or psychiatric disorders, such as anticonvulsantagents, as neuroprotective agents or for the management of pain. Theconantokins are administered to patients as described further below.

More specifically, the present invention is directed to conantokinpeptides, which include but are not limited to, G, T, L, R, S1, Oc, Gm,Ca2, Ca1 and Qu. Conantokin G (Con G) has the formulaGly-Glu-Xaa₁-Xaa₁-Leu-Gln-Xaa₂-Asn-Gln-Xaa₂-Leu-Ile-Arg-Xaa₂-Lys-Ser-Asn(SEQ ID NO:1), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid (Gla). The C-terminus contains a carboxyl or an amide, preferablyan amide group. Conantokin T (Con T) has the formulaGly-Glu-Xaa₁-Xaa₁-Tyr-Gln-Lys-Met-Leu-Xaa₂-Asn-Leu-Arg-Xaa₂-Ala-Glu-Val-Lys-Lys-Asn-Ala(SEQ ID NO:2), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid. The C-terminus contains a carboxyl or an amide, preferably anamide group. Conatokin L (Con L), has the formulaGly-Glu-Xaa₁-Xaa₁-Val-Ala-Lys-Met-Ala-Ala-Xaa₂-Leu-Ala-Arg-Xaa₂-Asp-Ala-Val-Asn(SEQ ID NO:3), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid. The C-terminus contains a carboxyl or an amide, preferably anamide group. Conantokin R (Con R) has the formula:Gly-Glu-Xaa₁-Xaa₁-Val-Ala-Lys-Met-Ala-Ala-Xaa₂-Leu-Ala-Arg-Xaa₂-Asn-Ile-Ala-Lys-Gly-Cys-Lys-Val-Asn-Cys-Tyr-Pro(SEQ ID NO:4), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid. The C-terminus contains a carboxyl or an amide, preferably acarboxyl group. The cysteine residues form a disulfide bridge.Conantokin S1 (Con S1) has the formula:Gly-Asp-Xaa₁-Xaa₁-Tyr-Ser-Lys-Phe-Ile-Xaa₂-Arg-Glu-Arg-Xaa₂-Ala-Gly-Arg-Leu-Asp-Leu-Ser-Lys-Phe-Pro(SEQ ID NO:5), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid. The C-terminus contains a carboxyl or amide, preferably a carboxylgroup. Conantokin Oc (Con Oc) has the formula:Gly-Glu-Xaa₁-Xaa₁-Tyr-Arg-Lys-Ala-Met-Ala-Xaa₁-Leu-Glu-Ala-Lys-Lys-Ala-Gln-Xaa₂-Ala-Leu-Lys-Ala(SEQ ID NO:6), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid. The C-terminus contains a carboxyl or amide, preferably an amidegroup. Conantokin Gm (Con Gm) has the formula:Gly-Ala-Lys-Xaa₁-Asp-Arg-Asn-Asn-Ala-Xaa₂-Ala-Val-Arg-Xaa₂-Arg-Leu-Glu-Glu-Ile(SEQI)NO:7),Xaa₁ and Xaa₂ are preferably γ-carboxyglutamic acid. The C-terminuscontains a carboxyl or amide, preferably an amide group. Conantokin Ca2(Con Ca2) has the formula:Gly-Tyr-Xaa₁-Xaa₁-Asp-Arg-Xaa₂-Ile-Ala-Xaa₂-Thr-Val-Arg-Xaa₂-Leu-Glu-Glu-Ala(SEQ ID NO:8), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid. The C-terminus contains a carboxyl or amide, preferably an amidegroup. Conantokin Qu (Con Qu) has the formula:Gly-Tyr-Xaa₁-Xaa₁-Asp-Arg-Xaa₂-Val-Ala-Xaa₂-Thr-Val-Arg-Xaa₂-Leu-Asp-Ala-Ala(SEQ ID NO:9), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid. The C-terminus contains a carboxyl or amide, preferably an amidegroup. Conantokin Ca1 (Con Ca1) has the formula:Gly-Asn-Asp-Val-Asp-Arg-Lys-Leu-Ala-Xaa₂-Leu-Xaa₂-Xaa₂-Leu-Tyr-Xaa₂-Ile(SEQ ID NO:68), wherein Xaa₂ is preferably γ-carboxyglutamic acid. TheC-terminus contains a carboxyl or amide, preferably an amide group.

The present invention is also directed to conantokin peptidederivatives. Examples of conantokin peptide derivatives includeconantokin peptides in which the γ-carboxyglutamic acid at the Xaa₂residues in these peptides is replaced by any other amino acids suchthat their NMDA antagonist activity is not adversely affected. Examplesof such replacements include, but are not limited to Ser, Ala, Glu andTyr. In addition, glutamic acid residues in the peptide can be modifiedto γ-carboxyglutamate residues. Other derivatives are produced bymodification of the amino acids within the conantokin structure.Modified amino acids include those which are described in Roberts et al.(1983). Other derivatives include conantokin peptides in which one ormore residues have been deleted.

The present invention is also directed to conantokin peptide chimeras.Suitable conantokin chimeras are produced by recombination of differentsegments of two or more conantokin peptides, conantokin peptidederivatives or a peptide encoded by exon 5 of the NMDA receptor, e.g.Lys-Pro-Gly-Arg-Lys (SEQ ID NO:10) or Lys-Pro-Gly-Arg-Lys-Asn (SEQ IDNO:11).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the time-dependent inhibition of audiogenic seizures by ConR following intracerebroventricular (i.c.v.) administration to Fringsaudiogenic mice.

FIG. 2 shows the ability of conantokins (Con R (♦), Con T (▪) Con G (◯))to block audiogenic seizures in a dose-dependent manner following i.c.v.administration to Frings audiogenic mice.

FIG. 3 shows the dose-dependent reduction in seizure severity followingi.c.v. administration to Frings audiogenic mice for conantokins (Con R(♦), Con T (▪) Con G (◯)).

FIG. 4 shows the dose-dependent blockage of audiogenic sequences by ConR at non-toxic doses. Protection (▪) and impairment (◯) for Con R areshown.

FIG. 5 shows the dose-response of the anticonvulsant activity of Con Gat one (□) and three () minutes following i.c.v. administration toFrings audiogenic mice.

FIG. 6 shows the dose-dependent inhibition of audiogenic seizuresfollowing i.c.v. (▪) or intravenous (i.v.) (◯) administration ofconantokin G.

FIG. 7 shows the time-dependent inhibition of audiogenic seizuresfollowing i.v. administration of conantokin G.

FIG. 8 shows the time-dependent inhibition of audiogenic seizuresfollowing per oral (p.o.) administration of conantokin G.

FIG. 9 shows the contralateral rotations following administration of 4mg/kg L-DOPA (▪), 4 mg/kg L-DOPA and 0.5 mM Con G () and 4 mg/kg L-DOPAand 5 mM Con G (▴) in a Parkinson's disease animal model.

FIG. 10 shows the ipsilateral rotations following administration of 4mg/kg L-DOPA (▪), 4 mg/kg L-DOPA and 0.5 mM Con G () and 4 mg/kg L-DOPAand 5 mM Con G (▴) in a Parkinson's disease animal model.

FIG. 11 shows the number of grooming episodes following administrationof SKF 38393 (▪) or the combination of SKF 38393 and Con G ().

FIG. 12 shows the number of net contralateral turns followingadministration of SKF 38393 (▪) or the combination of SKF 38393 and ConG (). The asterisks indicate statistical significance at p<0.05.

FIG. 13 shows the number of rearing episodes following administration ofSKF 38393 (▪) or the combination of SKF 38393 and Con G ().

FIG. 14 shows the number of cage lengths crossed followingadministration of SKF 38393 (▪) or the combination of SKF 38393 and ConG (). Statistical significance is shown at p<0.05 (*), p<0.01 (†) orp<0.01 (¥).

FIG. 15 shows the number of contralateral turns following administrationof SKF 38393 (▪) or the combination of SKF 38393 and Con G (). Theasterisks indicate statistical significance at p<0.05.

FIG. 16 shows the number of contralateral turns following administrationof SKF 38393 (▪) or the combination of SKF 38393 and Con G ().

FIG. 17 shows the effect of Con G (▴) and Con T (∇) on bladdercontraction amplitude.

FIG. 18 shows the effect of Con G (▴) and Con T (∇) on EUS EM Gactivity.

FIG. 19 shows the peptide stability of ECon G (▪), Con G (▴), Con R (▾)and Con T (♦) in 20% Frings mouse serum.

FIG. 20 shows the peptide stability of ECon G (▪), Con G (▴), Con R ()and Con T (□) in 20% Frings mouse liver homogenate.

FIG. 21 shows the stability of conantokin G in physiological saline atpH 6.0 at 37° C. (▪) and (▴).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is directed to conantokin peptides, conantokinpeptide derivatives and conantokin peptide chimeras, referred tocollectively as conantokins (unless the context dictates otherwise),having 10-30 amino acids, including preferably one to two or moreγ-carboxyglutamnic acid residues. The conantokins are useful for thetreatment of neurologic and psychiatric disorders, such asanticonvulsant agents, as neuroprotective agents or for the managementof pain, e.g. as analgesic agents. Neurologic disorders and psychiatricdisorders as used herein are intended to include such disorders asgrouped together in The Merck Manual of Diagnosis and Therapy, inclusiveof the disorders discussed herein.

More specifically, the present invention is directed to conantokinswhich are useful for the treatment and alleviation of epilepsy and as ageneral anticonvulsant agent. The present invention is also directed toconantokins which are useful for reducing neurotoxic injury associatedwith conditions of hypoxia, anoxia or ischemia which typically followsstroke, cerebrovascular accident, brain or spinal cord trauma,myocardial infarct, physical trauma, drowning, suffocation, perinatalasphyxia, or hypoglycemic events. The present invention is furtherdirected to conantokins useful for treating neurodegeneration associatedwith Alzheimer's disease, senile dementia, Amyotrophic LateralSclerosis, Multiple Sclerosis, Parkinson's disease, Huntington'sdisease, Down's Syndrome, Korsakoff's disease, schizophrenia, AIDSdementia, multi-infarct dementia, Binswanger dementia and neuronaldamage associated with uncontrolled seizures. The present invention isalso directed to conantokins which are useful for treating chemicaltoxicity, such as addiction, drug craving, alcohol abuse, morphinetolerance, opioid tolerance and barbiturate tolerance. The presentinvention is further directed to conantokins useful for treatingpsychiatric disorders, such as anxiety, major depression,manic-depressive illness, obsessive-compulsive disorder, schizophreniaand mood disorders (such as bipolar disorder, unipolar depression,dysthymia and seasonal effective disorder). The conantokins are alsouseful for treating ophthalmic disorders. The present invention is alsodirected to conantokins useful for treating additional neurologicaldisorders, such as dystonia (movement disorder), sleep disorder, musclerelaxation and urinary incontinence. In addition, the conantokins areuseful for memory/cognition enhancement, i.e., treating memory, learningor cognitive deficits. The present invention is also useful in thetreatment of HIV infection. Finally, the present invention is directedto conantokins useful for controlling pain, e.g. as analgesic agents,and the treatment of migraine, acute pain or persistent pain. They canbe used prophylactically and also to relieve the symptoms associatedwith a migraine episode.

The present invention is directed to conantokin peptides, which includebut are not limited to G, T, L, R, S1, Oc, Gm, Ca2, Ca1 and Qu.Conantokin G (Con G) has the formulaGly-Glu-Xaa₁-Xaa₁-Leu-Gln-Xaa₂-Asn-Gln-Xaa₂-Leu-Ile-Arg-Xaa₂-Lys-Ser-Asn(SEQ ID NO:1), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid (Gla). The C-terminus contains a carboxyl or an amide, preferablyan amide group. Conantokin T (Con T) has the formulaGly-Glu-Xaa₁-Xaa₁-Tyr-Gln-Lys-Met-Leu-Xaa₂-Asn-Leu-Arg-Xaa₂-Ala-Glu-Val-Lys-Lys-Asn-Ala(SEQ ID NO:2), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid. The C-termninus contains a carboxyl or an aamide, preferably anamide group. Conatokin L (Con L), has the formulaGly-Glu-Xaa₁-Xaa₁-Val-Ala-Lys-Met-Ala-Ala-Xaa₂-Leu-Ala-Arg-Xaa₁-Asp-Ala-Val-Asn(SEQ ID NO:3), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid. The C-terminus contains a carboxyl or an amide, preferably anamide group. Conantokin R (Con R) has the formula:Gly-Glu-Xaa₁-Xaa₁-Val-Ala-Lys-Met-Ala-Ala-Xaa₂-Leu-Ala-Arg-Xaa₂-Asn-Ile-Ala-Lys-Gly-Cys-Lys-Val-Asn-Cys-Tyr-Pro(SEQ ID NO:4), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid. The C-terminus contains a carboxyl or an amide, preferably acarboxyl group. The cysteine residues form a disulfide bridge.Conantokin S1 (Con S1) has the formula:Gly-Asp-Xaa₁-Xaa₁-Tyr-Ser-Lys-Phe-Ile-Xaa₂-Arg-Glu-Arg-Xaa₂-Ala-Gly-Arg-Leu-Asp-Leu-Ser-Lys-Phe-Pro(SEQ ID NO:5), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid. The C-terminus contains a carboxyl or amide, preferably a carboxylgroup. Conantokin Oc (Con Oc) has the formula:Gly-Glu-Xaa₁-Xaa₁-Tyr-Arg-Lys-Ala-Met-Ala-Xaa₂-Leu-Glu-Ala-Lys-Lys-Ala-Gln-Xaa₂-Ala-Leu-Lys-Ala(SEQ ID NO:6), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid. The C-terminus contains a carboxyl or amide, preferably an amidegroup. Conantokin Gm (Con Gm) has the formula:Gly-Ala-Lys-Xaa₁-Asp-Arg-Asn-Asn-Ala-Xaa₂-Ala-Val-Arg-Xaa₂-Arg-Leu-Glu-Glu-Ile(SEQ ID NO:7), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid. The C-terminus contains a carboxyl or amide, preferably an amidegroup. Conantokin Ca2 (Con Ca2) has the formula:Gly-Tyr-Xaa₁-Xaa₁-Asp-Arg-Xaa₂-Ile-Ala-Xaa₂-Thr-Val-Arg-Xaa₂-Leu-Glu-Glu-Ala(SEQ ID NO:8), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid. The C-terminus contains a carboxyl or amide, preferably an amidegroup. Conantokin Qu (Con Qu) has the formula:Gly-Tyr-Xaa₁-Xaa₁-Asp-Arg-Xaa₂-Val-Ala-Xaa₂-Thr-Val-Arg-Xaa₂-Leu-Asp-Ala-Ala(SEQ ID NO:9), wherein Xaa₁ and Xaa₂ are preferably γ-carboxyglutamicacid. The C-terminus contains a carboxyl or amide, preferably an amidegroup. Conantokin Ca1 (Con Ca1) has the formula:Gly-Asn-Asp-Val-Asp-Arg-Lys-Leu-Ala-Xaa₂-Leu-Xaa₂-Xaa₂-Leu-Tyr-Xaa₂-Ile(SEQ ID NO:68), wherein Xaa₂ is preferably γ-carboxyglutamic acid. TheC-terminus contains a carboxyl or amide, preferably an amide group.

The present invention is further directed to conantokin peptidederivatives. Examples of suitable derivatives include, but are notlimited to those described herein. In one embodiment, theγ-carboxyglutamic acid at the Xaa₂ residues in the above peptides may bereplaced by any other amino acids without adversely affecting their NMDAantagonist activity. Examples of such amino acid replacements include,but are not limited to, Ser, Ala, Glu and Tyr. In addition, glutamicacid residues in the peptide can be modified to γ-carboxyglutamateresidues. Substitutions of one amino acid for another can be made at oneor more additional sites within the above conantokin peptides, and maybe made to modulate one or more of the properties of the peptides.Substitutions of this kind are preferably conservative, i.e., one aminoacid is replaced with one of similar shape and charge. Conservativesubstitutions are well known in the art and include, for example:alanine to serine, arginine to lysine, asparagine to glutamine orhistidine, glycine to proline, leucine to valine or isoleucine, serineto threonine, phenylalanine to tyrosine, and the like. Other derivativesare produced by modification of the amino acids within the conantokinstructure. Modified amino acids include those which are described inRoberts et al. (1983). Other derivatives include conantokin peptides inwhich one or more residues have been deleted. For example, one suchderivative is conantokin G in which the five C-terminal amino acids havebeen deleted. The activity of such derivatives can easily be determinedin assays known in the art, including but not limited to the assaysdisclosed herein.

Finally, the present invention is directed to conantokin peptidechimeras. Suitable conantokin peptide chimeras are produced byrecombination of different segments of two or more conantokin peptides,conantokin peptide derivatives or the peptide encoded by exon 5 of theNMDA receptor, e.g. Lys-Pro-Gly-Arg-Lys (SEQ ID NO:10) orLys-Pro-Gly-Arg-Lys-Asn (SEQ ID NO:11). The conantokin peptides andconantokin peptide derivatives can be divided into, for example, fourdomains as shown in Table 1. Table 1 is not meant to be exclusive anddomains of conantokin peptides or conantokin peptide derivatives not setforth in Table 1 can also be easily identified. The SEQ ID NOs are inparentheses. “γ” is γ-carboxyglutamic acid.

TABLE 1 Domains of Conantokin Peptides and Derivatives Conantokin I IIIII IV Con G GEγγ (12) LQγNQγ (13) LIRγ (14) KSN Con T GEγγ (12) YQKMLγ(15) NLRγ (16) AEVKKNA (17) Con R GEγγ (12) VAKMAAγ (18) LARγ (19)NIAKGCKVNCYP (20) Con L GEγγ (12) VAKMAAγ (18) LARγ (19) DAVN (21)A^(7,10,14) Con G GEγγ (12) LQANQA (22) LIRA (23) KSN A⁷ Con G GEγγ (12)LQANQγ (24) LIRγ (14) KSN S⁷ Con G GEγγ (12) LQSNQγ (25) LIRγ (14) KSNT⁷ Con G GEγγ (12) LQTNQγ (26) LIRγ (14) KSN Con S1 GDγγ (27) YSKFIγ(28) RERγ (29) AGRLDLSKFP (30) Con Oc GEγγ (12) YRKAMAγ (31) LEAKKAQγ(32) ALKA (33) Con Qu GYγγ (34) DRγVAγ (35) TVRγ (36) LDAA (37) Con Ca2GYγγ (12) DRγIAγ (38) TVRγ (36) LEEA (39) Con Gm GAKγ (40) DRNNAγ (41)AVRγ (42) RLEEI (43) Con Ca1 GNDV (69) DRKLAγ (70) Lγγ LYγI (71)

The conantokin peptide chimeras are prepared by combining any one of theindividual elements of each domain with any one of the elements of theother domains. Thus, conatokin peptide chimeras are prepared bycombining (a) any one of domain I, (b) any one of domain II, (c) any oneof domain III and (d) any one of domain IV. Additional conantokinpeptide chimeras are prepared by combining domain I to the C terminalend of a peptide encoded by exon 5 of the NMDA receptor. Examples of thelatter peptide include Lys-Pro-Gly-Arg-Lys (SEQ ID NO:10) andLys-Pro-Gly-Arg-Lys-Asn (SEQ ID NO:1). The activity of such chimeras caneasily be determined in assays known in the art, including but notlimited to the assays disclosed herein.

In view of the definitions for conantokin peptides, conantokin peptidederivatives and conantokin peptide chimeras, a generic formula forconantokins of the present invention is derived. This generic formula isas follows:

(X₁)_(m)-G-X₂-X₃-X₄-(X₅)_(n)-(X₆)_(p)-(X₇)_(q)

wherein

X₁ is Lys-Pro-Gly-Arg-Lys (SEQ ID NO:10) or Lys-Pro-Gly-Arg-Lys-Asn (SEQID NO:11),

X₂ is any amino acid,

X₃ is any amino acid,

X₄ is any amino acid,

X₅ is a peptide having 1-7 amino acid residues,

X₆ is a peptide having 1-4 amino acid residues,

X₇ is a peptide having 1-12 amino acid residues,

m, n, p, and q are independently 0 or 1, with the proviso that when m is1, then n, p and q are each 0.

It is preferred that X₂ is Glu, Asp, Tyr, Ala or Asn, X₃ is Lys, Glu,Gla, Asp, Tyr, Ala, Ser or phosphoserine, X₄ is Glu, Gla, Asp, Ala, Seror phosphoserine and n is 1. More preferably, X₄ is Gla, and mostpreferably, X₃ and X₄ are each Gla.

The conantokin peptides, conantokin peptide derivatives, conantokinpeptide chimeras and conantokins of the generic formula above,collectively referred to as conantokins, have anticonvulsant activity inFrings audiogenic seizure susceptible mice and in syndrome-specificseizure animal models. These conantokins also have activity in animalpain models. These conantokins further have activity in in vitro assaysfor protection from neurotoxicity. These conantokins also have activityin animal models for Parkinson's disease. Thus, the conantokins of thepresent invention are useful as anticonvulsant agents, asneuroprotective agents, as analgesic agents, for managing pain and fortreating neurodegenerative disorders. The conantokins of the presentinventions are particularly useful as such agents for treatingneurologic disorders and psychiatric disorders that result from anoverstimulation of excitatory amino acid receptors. That is, theinvention pertains to disorders in which the pathophysiology involvesexcessive excitation of nerve cells by excitatory amino acids oragonists of the NMDA receptor(s). The conantokins are administered topatients as described further below.

These peptides, derivatives and chimeras are sufficiently small to bechemically synthesized. General chemical syntheses for preparing theforegoing conantokin peptides, conantokin peptide derivatives andconantokin peptide chimeras are described hereinafter, along withspecific chemical synthesis of one conantokin peptide and indications ofbiological activities of these synthetic products. Various ones of theconantokin peptides can also be obtained by isolation and purificationfrom specific Conus species using the technique described in U.S. Pat.No. 4,447,356 (Olivera et al., 1984), the disclosure of which isincorporated herein by reference.

Although the conantokin peptides of the present invention can beobtained by purification from cone snails, because the amounts ofconantokin peptides obtainable from individual snails are very small,the desired substantially pure conantokin peptides are best practicallyobtained in commercially valuable amounts by chemical synthesis usingsolid-phase strategy. For example, the yield from a single cone snailmay be about 10 micrograms or less of conantokin peptide. By“substantially pure” is meant that the peptide is present in thesubstantial absence of other biological molecules of the same type; itis preferably present in an amount of at least about 85% purity andpreferably at least about 95% purity. Chemical synthesis of biologicallyactive conantokin peptides depends of course upon correct determinationof the amino acid sequence.

The conantokin peptides can also be produced by recombinant DNAtechniques well known in the art. Such techniques are described bySambrook et al. (1979). The peptides produced in this manner areisolated, reduced if necessary, and oxidized to form the correctdisulfide bonds.

One method of forming disulfide bonds in the conantokin peptides of thepresent invention is the air oxidation of the linear peptides forprolonged periods under cold room temperatures or at room temperature.This procedure results in the creation of a substantial amount of thebioactive, disulfide-linked peptides. The oxidized peptides arefractionated using reverse-phase high performance liquid chromatography(HPLC) or the like, to separate peptides having different linkedconfigurations. Thereafter, either by comparing these fractions with theelution of the native material or by using a simple assay, theparticular fraction having the correct linkage for maximum biologicalpotency is easily determined. However, because of the dilution resultingfrom the presence of other fractions of less biopotency, a somewhathigher dosage may be required.

The peptides are synthesized by a suitable method, such as byexclusively solid-phase techniques, by partial solid-phase techniques,by fragment condensation or by classical solution couplings.

In conventional solution phase peptide synthesis, the peptide chain canbe prepared by a series of coupling reactions in which constituent aminoacids are added to the growing peptide chain in the desired sequence.Use of various coupling reagents, e.g., dicyclohexylcarbodiimide ordiisopropylcarbonyldimidazole, various active esters, e.g., esters ofN-hydroxyphthalimide or N-hydroxy-succinimide, and the various cleavagereagents, to carry out reaction in solution, with subsequent isolationand purification of intermediates, is well known classical peptidemethodology. Classical solution synthesis is described in detail in thetreatise, “Methoden der Organischen Chemie (Houben-Weyl): Synthese vonPeptiden,” (1974). Techniques of exclusively solid-phase synthesis areset forth in the textbook, “Solid-Phase Peptide Synthesis,” (Stewart andYoung, 1969), and are exemplified by the disclosure of U.S. Pat. No.4,105,603 (Vale et al., 1978). The fragment condensation method ofsynthesis is exemplified in U.S. Pat. No. 3,972,859 (1976). Otheravailable syntheses are exemplified by U.S. Pat. No. 3,842,067 (1974)and U.S. Pat. No. 3,862,925 (1975). The synthesis of peptides containingγ-carboxyglutamic acid residues is exemplified by Rivier et al. (1987),Nishiuchi et al. (1993) and Zhou et al. (1996).

Common to such chemical syntheses is the protection of the labile sidechain groups of the various amino acid moieties with suitable protectinggroups which will prevent a chemical reaction from occurring at thatsite until the group is ultimately removed. Usually also common is theprotection of an α-amino group on an amino acid or a fragment while thatentity reacts at the carboxyl group, followed by the selective removalof the α-amino protecting group to allow subsequent reaction to takeplace at that location. Accordingly, it is common that, as a step insuch a synthesis, an intermediate compound is produced which includeseach of the amino acid residues located in its desired sequence in thepeptide chain with appropriate side-chain protecting groups linked tovarious ones of the residues having labile side chains.

As far as the selection of a side chain amino protecting group isconcerned, generally one is chosen which is not removed duringdeprotection of the α-amino groups during the synthesis. However, forsome amino acids, e.g., His, protection is not generally necessary. Inselecting a particular side chain protecting group to be used in thesynthesis of the peptides, the following general rules are followed: (a)the protecting group preferably retains its protecting properties and isnot split off under coupling conditions, (b) the protecting group shouldbe stable under the reaction conditions selected for removing theα-amino protecting group at each step of the synthesis, and (c) the sidechain protecting group must be removable, upon the completion of thesynthesis containing the desired amino acid sequence, under reactionconditions that will not undesirably alter the peptide chain.

It should be possible to prepare many, or even all, of these peptidesusing recombinant DNA technology. However, when peptides are not soprepared, they are preferably prepared using the Merrifield solid-phasesynthesis, although other equivalent chemical syntheses known in the artcan also be used as previously mentioned. Solid-phase synthesis iscommenced from the C-terminus of the peptide by coupling a protectedα-amino acid to a suitable resin. Such a starting material can beprepared by attaching an α-amino-protected amino acid by an esterlinkage to a chloromethylated resin or a hydroxymethyl resin, or by anamide bond to a benzhydrylamine (BHA) resin or paramethylbenzhydrylamine(MBHA) resin. Preparation of the hydroxymethyl resin is described byBodansky et al. (1966). Chloromethylated resins are commerciallyavailable from Bio Rad Laboratories (Richmond, Calif.) and from Lab.Systems, Inc. The preparation of such a resin is described by Stewart etal. (1969). BHA and MBHA resin supports are commercially available, andare generally used when the desired polypeptide being synthesized has anunsubstituted amide at the C-terminus. Thus, solid resin supports may beany of those known in the art, such as one having the formulae—O—CH₂-resin support, -NH BHA resin support, or -NH-MBHA resin support.When the unsubstituted amide is desired, use of a BHA or MBHA resin ispreferred, because cleavage directly gives the amide. In case theN-methyl amide is desired, it can be generated from an N-methyl BHAresin. Should other substituted amides be desired, the teaching of U.S.Pat. No. 4,569,967 (Kornreich et al., 1986) can be used, or should stillother groups than the free acid be desired at the C-terminus, it may bepreferable to synthesize the peptide using classical methods as setforth in the Houben-Weyl text (1974).

The C-terminal amino acid, protected by Boc or Fmoc and by a side-chainprotecting group, if appropriate, can be first coupled to achloromethylated resin according to the procedure set forth in K. Horikiet al. (1978), using KF in DMF at about 60° C. for 24 hours withstirring, when a peptide having free acid at the C-terminus is to besynthesized. Following the coupling of the BOC-protected amino acid tothe resin support, the α-amino protecting group is removed, as by usingtrifluoroacetic acid (TFA) in methylene chloride or TFA alone. Thedeprotection is carried out at a temperature between about 0° C. androom temperature. Other standard cleaving reagents, such as HCl indioxane, and conditions for removal of specific α-amino protectinggroups may be used as described in Schroder & Lubke (1965).

After removal of the α-amino-protecting group, the remaining α-amino-and side chain-protected amino acids are coupled step-wise in thedesired order to obtain the intermediate compound defined hereinbefore,or as an alternative to adding each amino acid separately in thesynthesis, some of them may be coupled to one another prior to additionto the solid phase reactor. Selection of an appropriate coupling reagentis within the skill of the art. Particularly suitable as a couplingreagent is N,N′-dicyclohexylcarbodiimide (DCC, DIC, HBTU, HATU, TBTU inthe presence of HoBt or HoAt).

The activating reagents used in the solid phase synthesis of thepeptides are well known in the peptide art. Examples of suitableactivating reagents are carbodiimides, such asN,N′-diisopropylcarbodiimide andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide. Other activatingreagents and their use in peptide coupling are described by Schroder &Lubke (1965) and aKapoor (1970).

Each protected amino acid or amino acid sequence is introduced into thesolid-phase reactor in about a twofold or more excess, and the couplingmay be carried out in a medium of dimethylformamide (DMF):CH₂Cl₂ (1:1)or in DMF or CH₂Cl₂ alone. In cases where intermediate coupling occurs,the coupling procedure is repeated before removal of the α-aminoprotecting group prior to the coupling of the next amino acid. Thesuccess of the coupling reaction at each stage of the synthesis, ifperformed manually, is preferably monitored by the ninhydrin reaction,as described by Kaiser et al. (1970). Coupling reactions can beperformed automatically, as on a Beckman 990 automatic synthesizer,using a program such as that reported in Rivier et al. (1978).

After the desired amino acid sequence has been completed, theintermediate peptide can be removed from the resin support by treatmentwith a reagent, such as liquid hydrogen fluoride or TFA (if using Fmocchemistry), which not only cleaves the peptide from the resin but alsocleaves all remaining side chain protecting groups and also the α-aminoprotecting group at the N-terminus if it was not previously removed toobtain the peptide in the form of the free acid. If Met is present inthe sequence, the Boc protecting group is preferably first removed usingtrifluoroacetic acid (TFA)/ethanedithiol prior to cleaving the peptidefrom the resin with HF to eliminate potential S-alkylation. When usinghydrogen fluoride or TFA for cleaving, one or more scavengers such asanisole, cresol, dimethyl sulfide and methylethyl sulfide are includedin the reaction vessel.

Cyclization of the linear peptide is preferably affected, as opposed tocyclizing the peptide while a part of the peptido-resin, to create bondsbetween Cys residues. To effect such a disulfide cyclizing linkage,fully protected peptide can be cleaved from a hydroxymethylated resin ora chloromethylated resin support by ammonolysis, as is well known in theart, to yield the fully protected amide intermediate, which isthereafter suitably cyclized and deprotected. Alternatively,deprotection, as well as cleavage of the peptide from the above resinsor a benzhydrylamine (BHA) resin or a methylbenzhydrylamine (MBHA), cantake place at 0° C. with hydrofluoric acid (HF) or TFA, followed byoxidation as described above.

The peptides are also synthesized using an automatic synthesizer. Aminoacids are sequentially coupled to an MBHA Rink resin (typically 100 mgof resin) beginning at the C-terminus using an Advanced Chemtech 357Automatic Peptide Synthesizer. Couplings are carried out using1,3-diisopropylcarbodimide in N-methylpyrrolidinone (NMP) or by2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) and diethylisopropylethylamine (DIEA). The FMOC protecting groupis removed by treatment with a 20% solution of piperidine indimethylformamide(DMF). Resins are subsequently washed with DMF (twice),followed by methanol and NMP.

The conantokins are antagonists of the NMDA receptor subunits and areuseful as anticonvulsant agents, as neuroprotective agents, as analgesicagents, for managing pain and for treating neurodegenerative disorders.The conantokins of the present inventions are particularly useful assuch agents for treating neurologic disorders and psychiatric disordersthat result from an overstimulation of excitatory amino acid receptors.That is, the invention pertains particularly to disorders in which thepathophysiology involves excessive excitation of nerve cells byexcitatory amino acids or agonists of the NMDA receptor(s). Thus, theconantokins of the present invention are useful for the treatment andalleviation of epilepsy and as a general anticonvulsant agent. The useof conantokins in these conditions includes the administration of aconantokin in a therapeutically effective amount to patients in need oftreatment. The conantokins can be used to treat the seizures, to reducetheir effects and to prevent seizures.

The conantokins are also useful to reduce neurotoxic injury associatedwith conditions of hypoxia, anoxia or ischemia which typically followsstroke, cerebrovascular accident, brain or spinal chord trauma,myocardial infarct, physical trauma, drownings, suffocation,, perinatalasphyxia, or hypoglycemic events. To reduce neurotoxic injury, theconantokins should be administered in a therapeutically effective amountto the patient within 24 hours of the onset of the hypoxic, anoxic orischemic condition in order for the conantokins to effectively minimizethe CNS damage which the patient will experience.

The conantokins are further useful for the treatment of Alzheimer'sdisease, senile dementia, Amyotrophic Lateral Sclerosis, MultipleSclerosis, Parkinson's disease, Huntington's disease, Down's Syndrome,Korsakoff's disease, schizophrenia, AIDS dementia, multi-infarctdementia, Binswanger dementia and neuronal damage associated withuncontrolled seizures. The administration of the conantokins in atherapeutically effective amount to a patient experiencing suchconditions will serve to either prevent the patient from experiencingfurther neurodegeneration or it will decrease the rate at whichneurodegeneration occurs. In addition, the conantokins can beadministered in adjunct with conventional treatment agents to reduce theamount of such agents which need to be used.

The conantokins are also useful for treating chemical toxicity (such asaddiction, morphine tolerance, opiate tolerance, opioid tolerance andbarbiturate tolerance), anxiety, major depression, manic-depressiveillness, obsessive-compulsive disorder, schizophrenia, mood disorders(such as bipolar disorder, unipolar depression, dysthymia and seasonaleffective disorder), dystonia (movement disorder), sleep disorder,muscle relaxation, urinary incontinence, HIV infection and ophthalmicindications. In treating these conditions, a therapeutically effectiveamount of one or more conantokins is administered to a patient tocompletely treat the condition or to ease the effects of the condition.In addition, the conantokins are useful for memory/cognition enhancement(treating memory, learning or cognitive deficits), in which case atherapeutically effective amount of the conantokins is administered toenhance memory or cognition.

The conantokins are further useful in controlling pain, e.g., asanalgesic agents, and the treatment of migraine, acute pain orpersistent pain. They can be used prophylactically or to relieve thesymptoms associated with a migraine episode, or to treat acute orpersistent pain. For these uses, the conantokins are administered in atherapeutically effective amount to overcome or to ease the pain.

The anticonvulsant effects of conantokins have been demonstrated inanimal models. In rodents, conantokins are effective againstsupramaximal tonic extension seizures produced by maximal electroshockand threshold seizures induced by subcutaneous (s.c.) pentylenetetrazoleor picrotoxin. As described in further detail below, Conantokin R wasfound to have an antiseizure activity greater than 400,000-fold higherthan the standard commercial antiepileptic drug, valproic acid. Inaddition, Conantokin R was found to have a protective index at leasteight times better than that of valproic acid. Conantokins are alsoeffective against focal seizures induced by aluminum hydroxide injectioninto the pre- and post-central gyri of rhesus monkeys. Conantokins, whenadministered to patients with refractory complex partial seizures, maymarkedly reduce seizure frequency and severity. Thus, conantokins areuseful as anticonvulsant agents. Moreover, the clinical utility ofconantokins as a therapeutic agent for epilepsy may include generalizedtonic-clonic and complex partial seizures.

The neuroprotective effects of conantokins have been demonstrated inlaboratory animal models. Conantokins protected against hypoxic damageto the hippocampal slice in vitro. In neonate rats, conantokins reducedthe size of cortical infarcts and amount of hippocampal necrosisfollowing bilateral carotid ligation and hypoxia. Thus, conantokins areuseful as neuroprotective agents. Whereas other anticonvulsants mayexhibit neuroprotectant properties (Aldrete et al., 1979; Abiko et al.,1986; Nehlig et al., 1990), these effects often occurred only at high,clinically achievable doses associated with considerable toxicity(Troupin et al., 1986; Wong et al., 1986). In contrast, conantokinsexhibit both anticonvulsant and neuroprotectant effects at doses welltolerated by animals and humans.

The analgesic or anti-pain activity of conantokins is demonstrated inanimal models of pain and in animal models of persistent pain. In thesemodels, conantokins are (a) effective in nerve injury model studies; (b)effective in reducing the tolerance to opiate analgesics after chronicadministration and (c) effective in inhibiting activation of NMDAreceptors and thereby inhibiting the release of Substance P bysmall-diameter, primary, sensory pain fibers. Thus, conantokins areuseful as analgesic agents and anti-pain agents for the treatment ofacute and persistent pain. The conantokins are also useful for treatingaddiction, morphine/opiate/opioid tolerance or barbiturate tolerance.

The anti-neurodegenerative disease or neuroprotective activity ofconantokins is demonstrated in animal models of Parkinson's disease. Theconantokins are effective in reversing the behavioral deficits induce bydopamine depletion. The conantokins show behavioral potentiation,especially locomotor activity. The conantokins enhance the effect ofL-DOPA in reversing the behavioral deficits induce by dopaminedepletion. Thus, conantokins are effective neuroprotective agents andanti-neurodegenerative disease agents.

The effect of conantokins on muscle control is demonstrated in animals.At low doses, the conantokins are effective in hampering voiding at thelevel of the urethra. At higher doses, the conantokins are effective ineliminating all lower urinary tract activity. In the animal studies, itappears that the conantokins are more discriminatory in their inhibitoryeffects on striated sphincter than on bladder when compared with otherNMDA antagonists. Thus, the conantokins can be dosed in such a way so asto selectively decrease bladder/sphincter dyssynergia, especially inspinal cord injured patients, and are therefore useful for treatingurinary incontinence and muscle relaxation.

Pharmaceutical compositions containing a compound of the presentinvention as the active ingredient can be prepared according toconventional pharmaceutical compounding techniques. See, for example,Remington's Pharmaceutical Sciences, 17th Ed. (1985, Mack PublishingCo., Easton, Pa.). Typically, an antagonistic amount of activeingredient will be admixed with a pharmaceutically acceptable carrier.The carrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., intravenous, oral,parenteral or intrathecally.

For oral administration, the compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, lozenges, melts,powders, suspensions or emulsions. In preparing the compositions in oraldosage form, any of the usual pharmaceutical media may be employed, suchas, for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, suspending agents, and the like in thecase of oral liquid preparations (such as, for example, suspensions,elixirs and solutions), or carriers such as starches, sugars, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike in the case of oral solid preparations (such as, for example,powders, capsules and tablets). Because of their ease in administration,tablets and capsules represent the most advantageous oral dosage unitform, in which case solid pharmaceutical carriers are obviouslyemployed. If desired, tablets may be sugar-coated or enteric-coated bystandard techniques. The active agent must be stable to passage throughthe gastrointestinal tract. If necessary, suitable agents for stablepassage can be use and may include phospholipids or lecithin derivativesdescribed in the literature, as well as liposomes, microparticles(including microspheres and macrospheres).

For parenteral administration, the compound may dissolved in apharmaceutical carrier and administered as either a solution of asuspension. Illustrative of suitable carriers are water, saline,dextrose solutions, fructose solutions, ethanol, or oils of animal,vegetative or synthetic origin. The carrier may also contain otheringredients, for example, preservatives, suspending agents, solubilizingagents, buffers and the like. When the compounds are being administeredintracerebroventricularly or intrathecally, they may also be dissolvedin cerebrospinal fluid.

The conantokins can also be administered in a cell based delivery systemin which a DNA sequence encoding a conantokin is introduced into cellsdesigned for implantation in the body of the patient, especially in thespinal cord region. Suitable delivery systems are described in U.S. Pat.No. 5,550,050 and published PCT Application Nos. WO 92/19195, WO94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO96/40871, WO 96/40959 and WO 97/12635.

The conantokins are administered in an amount sufficient to antagonizethe effects of excitatory amino acids or other agonists upon the NMDAreceptor complex. The dosage range at which these conantokins exhibitthis antagonistic effect can vary widely depending upon the particulardisease or condition being treated, the severity of the patient'sdisease or condition, the patient, the specific conantokin beingadministered, the route of administration and the presence of otherunderlying disease states within the patient. Typically the conantokinsexhibit their therapeutic effect at a dosage range from about 0.015mg/kg to about 200 mg/kg, preferably from about 0.02 mg/kg to about 100mg/kg of the active ingredient, more preferably from about 0.03 mg/kg toabout 75 mg/kg of the active ingredient, and most preferably from about0.03 mg/kg to about 50 mg/kg of the active ingredient. A suitable dosecan be administered in multiple subdoses per day. Typically, a dose orsub-dose may contain from about 0.1 mg to about 500 mg of the activeingredient per unit dosage form. A more preferred dosage will containfrom about 0.5 mg to about 100 mg of active ingredient per unit dosageform.

For newly diagnosed patients with a seizure disorder and patients withseizure disorders for whom changes in drugs are being made, a relativelylow dosage of drug is started and increased over a week or so to astandard therapeutic dosage. After about a week at such dosage, bloodlevels are obtained to determine the patient's pharmacokinetic responseand, if appropriate, whether the effective therapeutic level has beenreached. If seizures continue, the daily dosage is increased by smallincrements as dosage rises above the usual. Once seizures are broughtunder control, the drug should be continued without interruption atleast one seizure-free year. At that time, discontinuation of the drugshould be considered, since about 50% of such patients will remainseizure free without drugs. Patients whose attacks initially weredifficult to control, those who failed a therapy-free trial and thosewith important social reasons for avoiding seizures should be treatedindefinitely.

EXAMPLES

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.The examples demonstrate the purification and chemical synthesis ofconantokin R. Conantokin Oc, conantokin S1, conantokin L, conantokin Gm,conantokin Ca1, conantokin Ca2 and conantokin Qu are purified andsynthesized in a similar manner. Conantokin derivatives and conantokinchimeras are made in conventional manner, such as chemicallysynthesizing the desired derivative or chimera.

Example 1 Methods

Sequence analysis. All sequence analyses were performed on the AppliedBiosystems Model 477A pulsed liquid phase automated protein sequencerusing the Nonnal-1 cycles. Peptides were immobilized on a glass fiberfilter treated with TFA, in the presence of 3 mg Polybrene and 0.2 mgNaCl. PTH-amino acid derivatives were analyzed by RP-HPLC with anon-line Applied Biosystems Model 120A analyzer, and identified byretention time.

Pyridylethylation. The peptide (20 to 300 pmoles) was dissolved in 50 μl0.25 M TRIS-HCl, 6 M in guanidine hydrochloride, and 2 mM in EDTA, pH7.5. Reduction was then carried out by adding 2 μl 10% mercaptoethanol,aq., and incubating at room temperature for 30 min. The peptide was thenalkylated by adding 2 μl of a 20% solution of 4-vinyl pyridine inethanol and incubating the mixture at room temperature, in the dark, for15 min. The solution was acidified prior to de-salting by RP-HPLC with 5μl of 10% TFA.

HPLC. Analytical and micro-preparative HPLC were performed on theHewlett-Packard Model 1090M system equipped with a diode array detector,using a Brownlee Aquapore RP-300 C-8 column, 2.1×100 mm and aTFA/acetonitrile system.

Amino acid analysis. Hydrolysis was performed in 4 N methanesulfonicacid at 110° C. for 24 hrs. The hvdrolysates were then analyzed on aPerkin-Elmer HPLC-based amino acid analyzer utilizing automatedpost-column OPA derivatization and fluorescence detection. Separation ofthe amino acids was done on an Interaction A-111 column at 55° C.

Total synthesis. Chemical synthesis of conantokin R and its C-terminalamidated analog was carried out essentially as described for conantokinG by Rivier et al. (1987), and as described further below. Mostfluorenylmethoxycarbonyl (Fmoc) amino acids were purchased from Bachem,Torrance, Calif. Fmoc-γ-γ-di-t-bu-γ-carboxyglutamic acid (Gla) wassynthesized as described by Rivier et al. (1987) Side chain protectionof Fmoc amino acids included Glu(γ-tbu), Lys(ε-Boc), Arg(MTR),Cys(S-trityl) and Tyr(O-but).

Example 2 Purification of Conantokin R

Specimens of Conus radiatus were collected by trawlers in Manila Bav.Venom ducts were dissected, and crude venom was harvested andfractionated using reverse phase HPLC chromatography as described underMethods. The various fractions were examined for sleep-inducingactivity. Several such fractions were identified, including a majorfraction referred to as the “sleeper-I peptide.” The sleeper-I peptidewas purified to homogeneity.

The amino acid sequence of the sleeper-I peptide was determined bystandard Edman methods. Two sequencing runs were carried out on thenative material. A number of blank positions were detected on the firstrun, because of the homology of the sequence obtained with previouslycharacterized conantokins T and G, we hypothesized that some of theblank positions might be γ-carboxyglutamate (Gla) residues which wouldbe consistent with a small of amount of Glu detected. In the secondsequencing run, the peptide was first reduced and alkylated in order toconvert cvsteines to the pyridylethylated derivative using4-vinylpyridine. Two of the blank positions (21 and 25) from the firstsequencing run yielded pyridylethylated Cys. This suggested that theremaining blank positions (3, 4, 11, 15) might be Gla residues.

Mass spectrometry was also used to assess the status of the missingresidues; electrospray mass spectrometry was used, yielding amonoisotopic mass (MH⁺) at mlz=3097.6. This value is consistent with theamino acid sequence if all remaining positions were indeedγ-carboxyglutamate residues, with the two cysteine residues in disulfidelinkage and a free carboxyl C-terminus (predicted monoisotopic mass is3097.4).

Together, the data are consistent with the structure of the sleeper-Ipeptide shown in SEQ ID NO:4, wherein the Cys residues are in disulfidelinkage. Because of the clear homology of the peptide to conantokins Gand T, this peptide was designated as conantokin R (for radiatus).

Example 3 Synthesis of Conantokin R and Conantokin R Amide

The proposed sequence assignment above was directly confirmed bychemical synthesis. Solid phase synthesis of both peptides on 3 g of theFmoc-Pro-palkoxybenzylalcohol resin and 1 g of the2,4-dimethoxybenzhydryl-amine resin (Rivier et al., 1987) was donemanually (0.5-3.0 mmol of amino acid/g of resin). Monitoring ofcoupling/deblocking for each cycle was done using the Kaiser test.(Kaiser, 1970) Removal of the Fmoc group was effected by treatment with0.1 M TBAF (tetrabutylammonium fluoride) in dimethylformamide (DMF) inthe case of the peptide acid and with two treatments (3+7) min of a 20%solution (v/v) of piperidine in DMF for the peptide amide. Resin washingwas accomplished by repeated application of DMF and/or dichloromethane(DCM) and methanol (MeOH). Couplings were mediated by1,3-diisopropylcarbodiimide (DIC) in DCM/DMF in the presence of 0.60.7eq of 1-hydroxybenzotiriazole (HOBt). Fmoc-Asn was incorporated into thepeptide with the side chain unprotected, in the presence of 0.6 to 2equivalent of HOBT in dimethylsulfoxide (DMSO)-DCM/DMF. The crudepeptides were purified using preparative HPLC techniques as previouslydescribed (Rivier et al., 1984). Peptides were dissolved in 0.1 M sodiumborate buffer in the presence of 10 mM CaCl₂ in order to obtain sharpabsorbances for CZE at pH 8.5 and HPLC at pH 7.4.

Conantokin R. Cleavage and deprotection of the peptide (2×2.0 gpeptido-resin) was achieved by treatment with a freshly prepared mixtureof trifluoroacetic acid, thioanisole, H₂O, ethanedithiol and DCM(40:8:1:2:49) (20 ml/g) at 37° C. for 7.25 hr. Trial cleavages of smallamounts (10 mg of peptide resin had demonstrated that the peptide wouldbe freed from the resin and all side chains deprotected, while the Glawould remain intact under these conditions. The peptide was precipitatedfrom the cleavage solution and washed by the addition of tert-butylmethyl ether (5×100 ml) in centrifuge tubes. The solid was suspended inwater (60 ml) and the suspension filtered. The tubes and resin werewashed with 40% acetic acid. The extracts were immediately cooled, andthen dissolved and diluted to 4 l (in the presence of 10 g ammoniumacetate pH 4.3). The pH was adjusted to 7.8 with NH₄OH and the solutionwas allowed to slowly stir and air oxidize at 4° C. for 3 d. Theprogress of oxidation was followed by the Ellman test and HPLC analysisas the oxidized product formed. After acidification of the solution topH 5 with acetic acid, the solution (2×≈1.5 l) was applied directly to apreparative HPLC cartridge. The gradient of acetonitrile applied to thepreparative cartridge in 0.1% TFA was 23-35% in 1 hr, with a flow rateof 100 ml/min. Analysis of the generated fractions was achieved usingisocratic conditions (24% acetonitrile, in 0.1% TFA) on a 5 μm Vydaccolumn. Peptide-containing fractions were then lyophilized to yield 120mg. The powder was re-applied to the preparative cartridge in TEAP pH2.3 and a gradient of acetonitrile 12-24% eluted the peptide. Desaltingwas carried out using an acetonitrile gradient from 23-35% in 0.1% TFAin 30 min, then re-application and elution using acetonitrile from12-57% in 10 min. Purified fractions were pooled and lyophilizedyielding conantokin R as the trifluoroacetate (36 mg). Results of theHPLC and capillary zone electrophoresis (CZE) analyses of this materialare given in Tables 1 and 2, respectively. Amino acid analysis afteracid hydrolysis gave the following ratios with expected values inparentheses: Asp (2), 1.96; Glu (5) including presence of four Glaresidues, 4.91; Pro (1) 1.26; Gly (2), 1.95; Ala (5) 5.00, Cys (2),1.65; Val (2) 1.97; Met (1), 0.88; Ile (1), 0.097; Leu (1), 1.01; Tyr(1), 0.98; Lys (3), 3.03; Arg (1), 0.98.

Conantokin R amide: Cleavage, deprotection and cyclization of thepeptide (1.6 g peptido-resin) was achieved as described above. However,after acidification of the oxidized solution to pH 5 with acetic acid,the solution was passed through two columns packed with cation-exchangeresin (2.5×7 cm) Bio-Rex 70 (H⁺ form). Eluent was checked by HPLC toverify absence of the peptide. The column was then washed with 1%aqueous acetic acid (250 ml) and the desired oxidized peptide was elutedwith 50% aqueous acetic acid. Fractions were collected (5 ml) and testedwith ninhydrin. Peptide-containing fractions were combined and diluted5× with H₂O, shell frozen and lyophilized (yield 300 mg). The gradientof acetonitrile applied to the preparative cartridge in TEAP pH 2.25 was15-30% in 1 hr, with a flow rate of 100 ml/min. Analysis of thegenerated fractions was achieved using isocratic conditions (21%acetonitrile in TEAP pH 2.25) on a 5 μm Vydac column. Peptide-containingfractions were then re-applied to the preparative cartridge in TEAP pH5.2 and the same gradient of acetonitrile applied. Desalting was carriedout using an acetonitrile gradient from 9-36% in 0.1% TFA in 45 min.Purified fractions were pooled and lyophilized yielding conantokin Ramide as the trifluoroacetate (12.5 mg). The purified material wasanalyzed by HPLC and capillary zone electrophoresis (see Tables 2 and3). Amino acid analysis of the synthetic material was consistent withthe conantokin R sequence. The synthetic material exhibited the samebiological activity as the native material, and a mixture of syntheticand native material gave a single homogeneous peak on HPLC.

A conantokin R analog with an amidated C-terminus was also synthesized.This material did not co-elute with the native conantokin R, verifyingthat in the natural peptide, the C-terminal Pro residue had a freecarboxyl group. Nevertheless, this material was also biologically activewith similar activity shown by conantokin R. Electrospray massspectrometry (MS) for conantokin R and conantokin R-amide, showedprotonated molecular ion [MH]⁺ at m/z=3097.6 and m/z=3096.7,corresponding to the calculated monoisotopic peptide acid of 3097.4 and3096.4, respectively. FAB MS was also performed for conantokin R and thespectrum showed an intact ion at m/z=3098.4.

TABLE 2 HPLC Analysis of Conantokin R (A) and Conantokin R-Amide (B)Solvent Flow Retention % System Rate Gradient Vol (ml) Purity A. TEAP pH7.4/CH₃CN^(a) 0.2 18-36% 2.0 85 CH₃CN in 30′ A. 0.1% TFA/CH₃CN^(b) 2.0 6-42% 54.0 ≈90 CH₃CN in 45′ B. TEAP pH 7.4/CH₃CN^(a) 0.2 18-36% 2.3 90CH₃CN in 30′ B. TEAP pH 2.3/CH₃CN^(b) 2.0 15-30% 31.0 90 CH₃CN in 20′^(a)UV monitoring at 214 nm, 0.12 absorbance unit at full scale. Columnwas Vydac (0.21 × 15 cm) packed with C₁₈ 5 μm particles, 300 Å poresize. ^(b)UV monitoring at 210 nm, 0.10 absorbance unit at full scale.Column was Vydac (0.46 × 25 cm) packed with C₁₈ 5 μm particles, 300 Åpore size.

TABLE 3 CZE Analysis of Conantokin R (A) and Conantokin R-Amide (B)Buffer Voltage Migration % System (kV) time (min) Purity A. 0.1 M sodiumborate in (85 15 9.0 ≈95 H₂O:15 CH₃CN) pH 8.5^(a) A. 0.1 M phosphate pH1.5^(b) 12 15.5 95 B. 0.1 M sodium borate in (85 15 8.4 ≈90 H₂O:15CH₃CN) pH 8.5^(a) B. 0.1 M phosphate pH 1.5^(b) 12 15.7 96 ^(a)UVmonitoring at 214 nm, 0.01 absorbance unit at full scale. Capillary wasBeckman eCAP fused silica (75 μm × 60 cm). Temperature was maintained at30° C. ^(b)UV monitoring at 214 nm, 0.01 absorbance unit at full scale.Capillary was Beckman eCAP fused silica (75 μm × 50 cm). Temperature wasmaintained at 30° C.

Example 4 Isolation of DNA Encoding Conantokins

DNA coding for the conantokins was isolated and cloned in accordancewith conventional techniques using the general procedures and probes orprimers set forth below.

Con G: The DNA was isolated using the toxin sequence degenerate probeDHOG108: CARGARAAYCARGARYT (SEQ ID NO:44) by Southern hybridization froma library of C. geographus DNA. The sequence of the DNA and itscorresponding amino acid sequence are set forth in SEQ ID NO:45 and SEQID NO:46, respectively. The mature peptide sequence prior to Glamodification (residues 81 to 97 of SEQ ID NO:46) contains a Val atposition 5 instead of Leu identified in the isolated peptide. TheC-terminal GKR are processed to a C-terminal amide in the maturepeptide.

Con R: The DNA was initially isolated using the probe DHOG424:CCYTTNGCDATRTTYTC (SEQ ID NO:47) by Southern hybridization from alibrary of C. radiatus DNA. The full length clone was obtained bySouthern Hybridization using the signal sequence probe DHOG450:GCCGTGCCTAGGATTA (SEQ ID NO:48). The sequence of the DNA and itscorresponding amino acid sequence are set forth in SEQ ID NO:49 and SEQID NO:50, respectively. The mature peptide sequence prior to Glamodification corresponds to residues 81 to 107 of SEQ ID NO:50.

Con Oc: The DNA was isolated using PCR with the signal sequence and 3′untranslated primers DHOG474: TGCTCGAATAAACATGAAAGATTTGGGGAA (SEQ IDNO:51) and DHOG475: TCTGCGATGCAACTGTACACGTATCTG (SEQ ID NO:52). Thesequence of the DNA and its corresponding amino acid sequence are setforth in SEQ ID NO:53 and SEQ ID NO:54, respectively. The mature peptidesequence prior to Gla modification corresponds to residues 74 to 96 ofSEQ ID NO:54. The C-terminal GR are processed to a C-terminal amide inthe mature peptide.

Con S1: The DNA was isolated using PCR with the signal sequence and 3′untranslated primers DHOG474 and DHOG475. The sequence of the DNA andits corresponding amino acid sequence are set forth in SEQ ID NO:55 andSEQ ID NO:56, respectively. The mature peptide sequence prior to Glamodification corresponds to residues 80 to 103 of SEQ ID NO:56.

Con L: The DNA was isolated using PCR with the signal sequence and 3′untranslated primers DHOG474 and DHOG475. The sequence of the DNA andits corresponding amino acid sequence are set forth in SEQ ID NO:57 andSEQ ID NO:58, respectively. The mature peptide sequence prior to Glamodification corresponds to residues 74 to 92 of SEQ ID NO:58. TheC-terminal GK are processed to a C-terminal amide in the mature peptide.

Con Gm: The DNA was isolated using PCR with the signal sequence and 3′untranslated primers DHOG474e: GGAATTCAATAAACATGAAAGATTTGGGGAA (SEQ IDNO:59) and DHOG475E: GGAATTCGCGATGCAACTGTACACGTATCTG (SEQ ID NO:60). Thesequence of the DNA and its corresponding amino acid sequence are setforth in SEQ ID NO:61 and SEQ ID NO:62, respectively. The mature peptidesequence prior to Gla modification corresponds to residues 74 to 92 ofSEQ ID NO:62. The C-terminal GKR are processed to a C-terminal amide inthe mature peptide.

Con Ca2: The DNA was isolated using PCR with the signal sequence and 3′untranslated primers DHOG474e and DHOG475e. The sequence of the DNA andits corresponding amino acid sequence are set forth in SEQ ID NO:63 andSEQ ID NO:64, respectively. The mature peptide sequence prior to Glamodification corresponds to residues 74 to 91 of SEQ ID NO:64. TheC-terminal GK are processed to a C-terminal amide in the mature peptide.

Con Qu: The DNA was isolated using PCR with the signal sequence and 3′untranslated primers DHOG474e and DHOG475e. The sequence of the DNA andits corresponding amino acid sequence are set forth in SEQ ID NO:65 andSEQ ID NO:66, respectively. The mature peptide sequence prior to Glamodification corresponds to residues 74 to 91 of SEQ ID NO:66. TheC-terminal GKRK are processed to a C-terminal amide in the maturepeptide.

On the basis of the peptide sequences for these conantokins, theconsensus N-terminal peptideMet-Xaa₁-Leu-Tyr-Thr-Tyr-Leu-Tyr-Leu-Leu-Val-Xaa₂-Leu-Val-Xaa₃-Xaa₄(SEQID NO:67), where Xaa₁ is His or Gln, Xaa₂ is Pro or Ser. Xaa₃ is Thr orAla and Xaa₄ is Leu or Phe is derived. Primers and/or probes are made onthe basis of this sequence and used alone or in combination with theprimers and/or probes described above to isolate additional conantokinpeptides from other species of Conus.

Example 5 Specificity of Conantokin R for NMDA Receptor Subtypes

Conantokins G and T were previously shown to inhibit NMDA receptors in avariety of systems. The efficacy of conantokin R was compared toconantokin G using cloned NMDA receptor subunit combinations expressedin oocytes.

The NR1 subunits can be functionally expressed as a homomeric NMDAreceptor complex in oocytes. From a comparison of the effects ofconantokin R and conantokin G on such homomeric NR1 subunit complex, itis clear that while conantokin G inhibits both of the major splicevariants tested, conantokin R is selective. At the concentrationstested, the peptide only inhibited the NR1.1 B subtype, with no effecton the corresponding A subtype. Specifically, Conantokin R at aconcentration of 3 μM inhibited approximately 95% of the currentproduced by the NR1-1b/NR2B NMDA subtype in response to glutamate andglycine. The use of lower concentrations of Conantokin R gave a K_(i) of0.14 μM for the NR1-1b/NR2B subtype. The affinity of Conantokin Rappears to be greater than 50-fold higher for this subtype than for theNR1-1b/NR2D combination, which was not affected even at 10 μM peptide.Conantokin R was found not to inhibit the AMPA receptor GluR1 andkainate receptors (GluR6).

The effect of conantokins G and R on heteromeric complexes containingboth NR1 and NR2 subunit combinations was also examined. In thecombinations of NR1:NR2B examined, the currents being elicited by thepresumptive heteromeric combination are much larger than when homomericNR1 subunits are expressed. Conantokin R and conantokin G both inhibitedsuch complexes if the B splice variant of the NR1 subunit was used.However, conantokin R proved to be selective for the B splice variant ofthe NR subunit, even in these heteromeric complexes, while conantokin Gwas not. The results indicate that conantokin R is a subtype-specificantagonist of the NMDA receptor, and has a preference for the B splicevariant which contains an additional 21 amino acids.

Example 6 In vivo Activity of Conantokins in Frings Audiogenic SeizureSusceptible Mice

In vivo anticonvulsant activity of conantokins was analyzed in Fringsaudiogenic seizure susceptible mice as described by White et al. (1992).The results for conantokin R are shown in Tables 4, 5 and 6.

TABLE 4 Effect of Conantokin R on the Audiodenic Seizure Susceptibilityof Frings Mice Following i.c.v. Administration Dose # Protected/# Tested# Protected/# Tested (pmol, i.c.v) 15 min. 60 min. 15 min. 60 min. 904/4 4/4 1/4 4/4 360 4/4 4/4 4/4 3/4 Ref: SW1:154

TABLE 5 Time Effect of Conantokin R Against Augiogenic SeizureSusceptibility of Frings Mice Following i.c.v. Administration Time (hrs)Dose 1/4 1/2 1 2 4 Reference # Prot./# Tested 9 pmol 2/4 2/3 3/4 1/4 0/4SW1:155, 159 # Toxic/# Tested 9 pmol 1/8 0/4 1/8 0/4 0/4 SW1:154, 160

TABLE 6 Effect of Conantokin R on the Audiogenic Seizure Susceptibilityof Frings Mice Following i.c.v. Administration # Protected/ # Toxic/Dose Seizure # Tested ED₅₀ # Tested TD₅₀ (pmol, i.c.v.) Score ± S.E.M.(at 1 hr) (pmol, i.c.v.) (at ¼ hr) (pmol) 2.27 5 ± 0 0/4 4.50  4.4 ±0.62 1/8 9 2.75 ± 0.86 4/8 9.00 1.8 1.125 ± 0.58  7/8 (5.98-14.3)* 9 0 ±0 4/4 1/8 164 18.0 — — 4/8 (111-233)* 36.0 0 ± 0 4/4 8/8 *95% confidenceinterval Ref: SW1:153, 154, 159-161

Conantokin R yielded an effective dose (ED₅₀) of 9 pmol. The ED₅₀ forconantokin-T was 5.1 pmol (95% CI=3.3-9.5 pmol). The ED₅₀ for conantokinG was 1.0 pmol (95% CI=1.0-2.0 pmol). Furthermore, conantokin R yieldeda toxic dose (TD₅₀) of 164 pmol. The dose required to elicitneurotoxicity was 18 times greater than the effective dose(TD₅₀/(ED₅₀—164/9=18=Protective Index, PI). The TD₅₀ for conantokin Gwas 28 pmol (95% CI=22-35 pmol), yielding a protective index of 27.Moreover, the PI of 18 for conantokin R and 27 for conantokin G exceedsthat of other anti-seizure medications tested in this model. Thetherapeutic dose of typical anti-seizure medications is close to thetoxic dose (typical PI=2-3). Since the protective index is high forconantokin R and conantokin G, these peptides will be better toleratedthan previous anti-convulsant agents.

Similar results are obtained for conantokin S1, G, T, L, Gm, Ca2 and Quand analogs of these peptides in which the γ-carboxyglutamic acidresidues other than at positions 3 and 4 are substituted by other aminoacid residues, including Ser, Ala, Glu and Tyr. These results areconsistent with the finding that several Con G synthetic analogs possesshigh affinities for non-competitive inhibition of polyamine enhanced[³H]MK-801 binding (Zhou et al., 1996).

Example 7 Comparison of In vivo Activity of Conantokins and Standards inFrings Audiogenic Seizure Susceptible Mice

The anticonvulsant profile of several conantokins and the standardsdizocilpine (MK-801), ifendropil and valproic acid was determined usingFrings audiogenic seizure-susceptible mice (25-30 g body weight)obtained from the house colony of the University of Utah. Allintracerebroventricularly (i.c.v.) injections were made free-handed intothe lateral ventricle (approximately 1 mm lateral, 1 mm anterior frombregma and to a depth of 3 mm from the surface of the skull) of awakemice with a 10 μl Hamilton syringe. Varying doses of the compounds weretested. At the predetermined time of peak anticonvulsant effect,individual mice were placed into a round Plexiglas chamber (diameter, 15cm; height, 18 cm) pitted with an audio transducer (Model A5-ZC; FETResearch & Development, Salt Lake City, Utah) and exposed to a highintensity sound stimulus (110 decibels, 11 KHz) for 25 seconds. Animalsnot displaying tonic forelimb or hindlimb extension were consideredprotected. The effect of the test compounds on motor performance wasassessed by the rotorod test (Dunham and Miya, 1957). For thisprocedure, mice were tested for their ability to maintain balance on arotating (6 rpm) knurled Plexiglas rod (1 inch diameter) for one minute.Mice unable to maintain balance in three successive trials during thetest period were considered toxic. The median effective dose (ED₅₀) andthe median toxic dose (TD₅₀) was calculated by probit analysis (Finney,1971). For these studies, the dose of each test substance was variedbetween the limits of 0 and 100% protection and toxicity. The protectiveindex (PI) is TD₅₀/ED₅₀. The results are shown in Tables 7 and 8. Thetime-dependent inhibition of audiogenic seizures by Con R followingi.c.v. administration is shown in FIG. 1. The ability of conantokins(Con R (♦), Con T (▪) Con G (◯)) to block audiogenic seizures in adose-dependent manner following i.c.v. administration is shown in FIG.2. The dose-dependent reduction in seizure severity following i.c.v.administration for conantokins (Con R (♦), Con T (▪) Con G (◯)) is shownin FIG. 3. The dose-dependent blockage of audiogenic seizures by Con Rat non-toxic doses is shown in FIG. 4. Protection (▪) and impairment (◯)are shown with an ED₅₀ of 9 pmol and a TD₅₀ of 164 pmol.

TABLE 7 Comparative Anticonvulsant Efficacy, Minimal Motor Impairmentand Protective Index of Conantokins R, T, and G and Ifenprodil FollowingI.C.V. Administrations Test Time of Test^(a) nmols, i.c.v. Substance(min) ED₅₀ ^(b) TD₅₀ ^(b) P.I. Con R 60, 15  0.013^(c)  0.228^(d) 17(0.0083-0.020)  (0.154-0.323) Con T 30, 15 0.017 0.228 13 (0.011-0.032)(0.154-0.323) Con G 30, 15 0.0035 0.094 27 (0.002-0.005) (0.073-0.1 16)Ifenprodil  5, 5 ˜25 <25 <1 ^(a)First time, ED₅₀; Second time, TD₅₀^(b)95% confidence interval in parentheses ^(c)The values for ED₅₀ andTD₅₀ for Con R, Con T, and Con G are the raw data. These numbers aremultiplied by 0.72 for Con R and by 0.3 for Con G and Con T to obtainvalues corrected for peptide content. The PI numbers do not change.

TABLE 8 Comparative Anticonvulsant Efficacy, Minimal Motor Impairmentand Protective Index of Conantokin R, MK801, Ifenprodil and ValproicAcid Following I.C.V. Administration Test Time of Test^(a) nmols, i.c.v.Substance (min) ED₅₀ ^(b) TD₅₀ ^(b) P.I. Con R 60, 15  0.013^(c) 0.228^(d) 17 (0.0083-0.020)  (0.154-0.323) MK801 5, 5 0.641 1.227 1.9(0.415-0.933) (0.639-4.532) Valproic 5, 5 5644 >6000 <12,000 1.1-2.2Acid (3707-7759) Ifenprodil 5, 5 ˜25 <25 <1 ^(a)First time, ED₅₀; Secondtime, TD₅₀ ^(b)95% confidence interval in parentheses ^(c)The values forED₅₀ and TD₅₀ for Con R are the raw data. These numbers are multipliedby 0.72 for Con R to obtain values for peptide content. The PI numberdoes not change.

Example 8 In vivo Activity of Conantokins in CF No. 1 Mice

In vivo anticonvulsant activity of conantokins R, S1, G, T, L, Gm, Caand Qu are analyzed in CF No. 1 mice as described by White et al.(1995), using the maximal electroshock, subcutaneous pentylenetetrazole(Metrazol) seizure threshold and threshold tonic extension test. Each ofthe conantokins tested are found to have anticonvulsant activity.Specifically, the activity of Conantokins R and G in this model animalare shown in Table 9.

TABLE 9 Anticonvulsant Efficacy, Minimal Motor Impairment and ProtectiveIndex of Conantokins G and R Following I.C.V. Administration Test FringsAudiogenic Mice CF #1 Mice Substance ED₅₀ ^(a) TD₅₀ ^(a) P.I.^(b) MESED₅₀ ^(a) TD₅₀ ^(a) P.I.^(b) Con G 0.0035^(c) 0.094^(c) 27 0.026 0.0662.5 (0.002-0.005) (0.073-0.116) (0.013-0.038) (0.048-0.091) Con R 0.0130.228 17 0.083 ˜0.300 3.6 (0.0083-0.020) (0.154-0.323) (0.029-0.117)^(a)nmols; 95% confidence intervals in parentheses ^(b)Protective Index(TD₅₀/ED₅₀) ^(c)The values for ED₅₀ and TD₅₀ for Con R and Con G are theraw data. These numbers are multiplied by 0.72 for Con R and by 0.3 forCon G to obtain values corrected for peptide content. The PI numbers donot change.

Example 9 In vivo Activity of Conantokin T in Frings AudiogenicAudiogenic Seizure-Susceptible Mice Following I.V. Administration

In vivo anticonvulsant activity of Con T was analyzed in Fringsaudiogenic seizure-susceptible mice as described above except that thepeptide was administered intravenously (IV) at 12 mg/kg. The peptide wasadministered to naive mice and pre-stimulated mice. The mice were dosedi.v. and stimulated at the indicated time intervals and the protectionwas measured. The pre-stimulated mice were stimulated at 1 minute as apre-stimulation and then stimulated at the indicated time intervals. Theresults are shown in Tables 10 and 11. No animals exhibited behavioraltoxicity at this dose, as determined by the rotorod test as describedabove.

TABLE 10 Anticonvulsant (Frings Audiogenic Seizure-Susceptibile MouseModel) Activity of Conantokin T Following Intravenous (IV)Administration: Naive Animals Con-T Saline Control Time of Test #Protected/ # Protected/ (min) # Tested % Protected # Tested % Protected1 10 0/9  0% 0/5 0% 20 1/2 50% 0/2 0% 30 2/6 33% 0/5 0% 60 4/6 67% 2401/6 17%

TABLE 11 Anticovulsant (Frings Audiogenic Seizure-Susceptible MouseModel) Activity of Conantokin T Following Intravenous (IV)Administration: Pre-Stimulated Animals Con-T Saline Control Time of Test# Protected/ # Protected/ (min) # Tested % Protected # Tested %Protected 1 1/2 50% 0/2 0% 10 20 1/2 50% 0/2 0% 30 3/5 60% 60 6/7 86% 6/12 50%  240  6/11 55% 0/4 0%

Table 10 shows 67% protection for the naive animals at 60 minutesfollowing the i.v. of Conantokin T. The pre-stimulation sometimesresults in erratic protection which may be to compromising theblood-brain-barrier, thus, allowing CNS penetration by compoundsotherwise would not penetrate. Alternatively, the result in thepre-stimulated animals could be due to making the animals refractory tosubsequent seizures. Nevertheless, the present experiment demonstratesthe bioavailability of Conantokin T, since it protected the naiveanimals following i.v. dose. Similar results were obtained withConantokin G.

Example 10 In vivo Activity of Conantokin G in Frings AudiogenicSeizure-Susceptible Mice Following I.C.V. Administration

In vivo anticonvulsant activity of conantokin G was analyzed in Fringsaudiogenic seizure susceptible mice as described above, except that thepeptide was administered i.c.v. at 0.0038 nmol or 0.0056 nmol. Table 12shows the time to onset of the anticonvulsant activity of Con Gfollowing i.c.v. administration.

TABLE 12 Time to Onset of Anticonvulsant Activity of Conantokin G:Seizure Protection Following I.C.V. Administration to Frings AudiogenicMice Percent Protection Test Dose (at time of test, min) Substance nmol,i.c.v. 1 2 3 4 5 60 Con G¹ 0.0038 0 0 0 100 100 87.5 Con G² 0.0056 25 50100 — — 100 ¹N = 16; ²N = 8

At a Con G dose of 0.038 nnol, seizure protection was observed in 100%of the animals tested at four minutes. As a control, at the same dose ofCon G, 87.5% of the animals were protected at 60 minutes. Moreover, at aCon G dose of 0.0056 nmol, seizure protection was observed in 25% of theanimals tested at one minute, 50% at two minutes and 100% at threeminutes. As a control, at the same dose of Con G, 100% of the animalswere protected at 60 minutes. No animals exhibited behavioral toxicity(rotorod minimal motor impairment) at the doses of Con G and the timestested. Thus, Con G elicits a very rapid time to onset (within one tothree minutes) of anticonvulsant activity, with high potency and lowbehavioral toxicity, following i.c.v. administration to Fringsaudiogenic mice.

A dose-response of the anticonvulsant activity of Con G at one and threeminutes following i.c.v. administration in this model is shown in FIG.5. These data demonstrate that the median effective dose (ED₅₀) foranticonvulsant activity of Con G at one and three minutes was 0.023 nmol0.004 mnol, respectively.

Example 11 In vivo Activity of Conantokin G in Frings AudiogenicSeizure-Susceptible Mice Following I.C.V. Administration

In vivo anticonvulsant activity of Conantokin G was analyzed in Fringsaudiogenic seizure susceptible mice as described above with i.c.v.administration. Table 13 compares the median effective dose (ED₅₀), themedian toxic dose (TD₅₀), rotorod minimal motor impairment andprotective index (PI=TD₅₀ED₅₀) of Con G at one, three and thirty minutesfollowing i.c.v. administration. In these studies, all rotorod minimalmotor impairment tests were performed at 30 minutes.

TABLE 13 Time to Onset of Anticonvulsant Activity of Conantokin G:Efficacy, Minimal Motor Impairment and Protective Index Following I.C.V.administration to Frings Audiogenic Mice Test Time of Dose (nmol,i.c.v.) Substance Test (min) ED₅₀ TD₅₀ PI Con G  1^(1,2) 0.023 0.028 1.2(0.022-0.035)³ Con G 3 0.004 0.028 7.8 (0.003-0.004) (0.022-0.035) Con G30  0.001 0.028 27 (0.001-0.002) (0.022-0.035) ¹N = 8 animals/group ²Noanimals exhibited minimal motor impairment at indicated doses at thetime of test. ³95% confidence interval in parentheses.

At one minute following Con G administration, the ED₅₀, TD₅₀ and PI foranticonvulsant activity were 0.023 nmol, 0.028 nmol and 1.2,respectively. At three minutes following Con G administration, the ED₅₀,TD₅₀ and PI for anticonvulsant activity were 0.004 nmol, 0.028 nmol and7.8, respectively. At 30 minutes following Con G administration, theED₅₀, TD₅₀ and PI for anticonvulsant activity were 0.001 nmol, 0.028nmol and 27, respectively. These data clearly show that the time toonset of Con G anticonvulsant activity following i.c.v. administrationto Frings audiogenic mice was very rapid (within one to three minutes)with very low behavioral toxicity compared to prototypical antiseizuredrugs in testing or on the market.

Example 12 In vivo Phencyclidine-Like Behavioral Effects of Conantokin GFollowing I.C.V. Administration

The in vivo phencyclidine-like behavioral effects of Con G was assessedby the elevated platform test as described by Evoniuk et al. (1991). Theplatform test is a rapid method for evaluating the behavioral effects ofphencyclidine-like dissociative anesthetics in mice. At 15 minutesfollowing a Con G dose of 0.0225 nmol (i.c.v.) to mice, no drug-inducedfalls from the elevated platform were observed. Alternatively, as acontrol, a 44.5 nmol dose of MK 801 (dizocilpine) elicited 87.5%drug-induced falls from the elevated platform. Thus, Con G does notinduce phencyclidine-like behavioral effects in mice. The results areshown in Table 14.

TABLE 14 Absence of Phencyclidine-Like Behavioral Effects UsingConantokin G Compared to MK 801: Activity in the Elevated Platform TestTest Maximum Dose Percent Drug-Induced Substance Tested (nmol) Fallsfrom Elevated Platform Con G 0.0225¹ (i.c.v.) 0 MK 801 44.5² (i.p.) 87.5MK 801 118.6 (i.p.) 100 H₂O 50 μl (i.p.) 0 ¹ED₅₀, TD₅₀, PI as noted inExample 11. ²Dose used in these studies was the same as the minimumeffective dose that induced ≧50% of animals to fall from elevatedplatform in Evoniuk et al. (1991).

Example 13 Comparison of Modes of Administration of Conantokin G

In vivo anticonvulsant activity of conantokin G when administeredi.c.v., i.v. or p.o. was analyzed in Frings audiogenic mice as describedabove. The ED₅₀ at 30 minutes was determined to be 0.048 nmol/kg for thei.c.v. administration and 702 nmol/kg for the i.v. administration. The95% confidence interval for these values are 0.027-0.072 for i.c.v.administration and 341-1246 for i.v. administration. FIG. 6 shows thedose-dependent inhibition of audiogenic seizures for i.c.v. and i.v.administration of Con G. The time-dependent inhibition of audiogenicseizures by Con G when administered i.v. (2650 nmol/kg) or p.o. (6623nmol/kg) is shown in FIGS. 7 and 8, respectively.

Example 14 Comparison of In Vivo Activity of Conantokin G with PrototypeAntiepileptic Drugs

The in vivo anticonvulsant activity of Con G was compared to theanticonvulsant activities of several prototype antiepileptic drugs (AED)with i.c.v. administration in Frings audiogenic mice, as describedabove. The results are shown in Table 15. The protective index (PI) ofCon G was significantly higher than the PI for the other drugs tested.

TABLE 15 Effect of Conantokin G Compared to Prototype AntiepilepticDrugs (AED) on Audiogenic Seizure-Sesceptibility Following i.c.v.Administration to Frings Mice Prototype Time of Test ED₅₀ TD₅₀ AEDs(min)* nmol nmol PI Con G 30, 15  0.001 0.028 27 (0.001-0.002)(0.022-0.035) Con G 3, 15 0.004 0.028 7.8 (0.003-0.004) (0.022-0.035)Phenobarbitol 5, 15 145 68.8 0.5 (105-186) (42.9-93.5) Valproic Acid 5,5  566 >6000- 1.1-2.2 (3707-7759) <12,000 Lamotrigine 30, 15-60146 >290 >2.0 (101-195) limit of solubility Felbamate 30, 15-60 5/8protected at 525 no marked toxicity unable to 4/8 protected at 630 up to630 determine Topiramate 15-240, >150 nmol no marked toxicity unable to15-240 limit of solubility determine TD₅₀ = rotrod performance, minimalmotor impairment measure of behavioral toxicity 95% confidence intervalin parenthesis PI = Protective Index (PI =TD₅₀/ED₅₀) * = 1st time ED₅₀;2nd time TD₅₀

Example 15 In Vivo Activity of Conantokin G inPentylenetetrazole-Induced Threshold Seizure Model

The in vivo activity of Con G was analyzed using timed intravenousinfusion of pentylenetetrazole (White et al., 1995). At time to peakeffect, the convulsant solution (0.5% pentylenetetrazole in 0.9% salinecontaining 10 U.S.P. units/ml heparin sodium) is infused into the tailvein at a constant rate of 0.34 ml/min. The time in seconds from thestart of the infusion to the appearance of the first twitch and theonset of clonus is recorded for each drug treated or control animal. Thetimes to each endpoint are converted to mg/kg of pentylenetetrazole foreach mouse, and mean and standard error of the mean are calculated. Theresults are shown in Table 16. Administration of Con G i.c.v. at 18.75pmol elevates the i.v. pentylenetetrazole seizure threshold.

TABLE 16 Conantokin G Elevates i.v. Pentylenetetrazole (PTZ) Seizurethreshold PTZ, mg/kg Treatment Dose, pmol First Twitch Clonus Control 029.9 ± 2.5 41.9 ± 4.8 Conantokin G 18.75 47.3 ± 7.8  75.8 ± 11.1 N = 7(Control, 8 (Conantokin G)

Example 16 In Vivo Activity of Conantokin G in Parkinson's DiseaseAnimal Model

The anti-Parkinsonian potential of conantokin G was examined in ratswith unilateral lesions of the nigrostriatal dopamine system. Theunilateral lesions are created by local infusion of the neurotoxin6-hydroxydopamine (6-OHDA) into the right substantia nigra ofanesthetized rats. The rats recovered for two weeks at which time theyare anesthetized and guide cannulae implanted into the brain, ending inthe right lateral ventricle. The guide cannulae are kept patent with astylet placed in the guide cannula. One week later, the rats are placedin a cylindrical Plexiglas® cage, the stylet is removed, and an infusioncannula is inserted into the guide. The infusion cannula is attached toa syringe on an infusion pump which delivered conantokin G (0.5 mM or5.0 mM) or control vehicle at a rate of 1 μl/min for a total injectionof 2 μl (1 nmol/2 μl). Fifteen minutes after the injection of conantokinG, L-Dopa (4 mg/kg ip) is injected. The number of full rotationscontralateral and ipsilateral to the dopamine-depleted hemisphere isthen counted for 2 minutes, every 10 minutes, for 2 hours. A video ofthe rats is also made to follow the behavioral potentiation of thetreatment. The results are shown in FIGS. 9 and 10. These results showthat there is clear potentiation of the L-Dopa activity with Con G. Thevideo showed that the behavioral potentiation by Con G is very dramatic,especially the locomotor activity. The ability to elicit contralateralrotation in this animal model leads to the conclusion that the testedcompounds reverse the behavioral deficits induced by dopamine depletion.In addition to the above tests, the in vivo activity of Con G incombination with SKF 38393 was compared with that of SKF 38393 alone.The results are shown in FIGS. 11-16. The combination of Con G and SKF38393 demonstrated increased activity.

Example 17 Regulation of Striatal Output Pathways by NMDA-2B Receptors

Experimental Parkinsonism results in altered functional activity ofstriatal output pathways. Neurons projecting to the globus pallidus(indirect pathway) become overactive, resulting in increased inhibitionof cortical regions involved in movement and movement initiation viaprocessing through the basal ganglia-thalamo-cortical loop. Treatmentwith non-selective NMDA antagonists (Klockgether and Turski, 1990) orlesions of the glutamergic neurons of the subthalamic nucleus alleviateParkinsonian symptoms in experimental models (Bergman et al., 1990). Theidentification of multiple subtypes of NMDA receptor subunits and theirdifferential expression throughout the basal ganglia nuclei offers thepotential of altering glutamatergic transmission with specific nuclei.Of the primary basal ganglia nuclei, the NR2B subunit is expressedalmost exclusively in their striatum (Standaert et al., 1994).

The recent discovery of a class of conantokins with remarkableselectivity for NDMA receptors expressing the NR2B subunit offers aunique pharmacological tool for the investigation of the role of thisNMDA receptor subtype in the regulation of basal ganglia circuits, andits potential as a target for the treatment of movement disorders suchas Parkinsonism. Con R will be administered i.c.v. alone or incombination with SKF 38393 in unilateral 6-hydroxydopamine-lesionedrats, or in combination with eticlopride in uniesioned rats, toprecisely examine the role of NR2B receptors on immediate early geneinduction in striatonigral and striatopallidal neurons whichspecifically express dopamine DI or D2 receptors, respectively

Example 18 In Vivo Activity of Conantokin G in Animal Model of UrinaryIncontinence

Female Wistar rats are anesthetized with urethane and, followingtracheotomy (for ventilation after skeletal muscle paralysis) andjugular and carotid cannulation (for drug delivery and blood pressurerecording, respectively), laminectomies are performed at C7-T2 andT11-S1 through a midline dorsal incision. The back is temporarily closedand the animal is placed abdomen up. A midline incision is made from thesternum to the pubis. The ureters are isolated, ligated and cutproximally, and saline soaked gauze wicks are positioned at the cut endto exit the abdominal incision for urine elimination.

A double lumen urethral catheter is passed through a cystotomy at thedome of the bladder and seated in the urethral opening at the level ofthe internal sphincter. A second, single lumen catheter is positioned,through its own cystotomy, into the bladder. Both catheters are tied inwith suture, and connected to pressure recording transducers andfilling/perfusion syringes via three-way stopcocks.

Following closing of the abdomen, the rat is placed onto its abdomen andthe back incision reopened and sutured to a metal ring to form a pocketfor oxygenated Krebs solution. The spinal cord is cut at C7 and L3.Silver wire electrodes are introduced into the cut ends at C7 (insertedcaudally) and L3 (inserted rostrally) for sympathetic preganglionicstimulation. Spinal roots L6 and S1 are placed on a hook electrode forstimulation of parasympathetic preganglionic axons. A continuouslyoxygenated Krebs solution fills the dorsal pocket and bathe the spinalcord and roots.

Simultaneous, independent electrical stimulation is delivered a lowlevels to both sets of preganglionic pathways and independent adjustmentof stimulus parameters is made to achieve maximal responses from theblood pressure (sympathetic preganglionic stimulus driven) and thebladder (parasympathetic preganglionic driven).

Conantokins are introduced intrathecally and changes in blood andbladder pressure responses under constant drive are monitored, recordedand taped. Control studies are made with hexamethonium bromide and allstudies finish with hexamethoniun bromide administration.

Conatokin G was administered intrathecally at 0.3 nmol, 3.0 nmol and 30nmol. The 0.3 nmol dose had no effect. The 3.0 nmol dose had no effecton bladder contraction amplitude, but increased frequency. This meansthat voiding was hampered at the level of the urethra. The 30 nmol doseeliminated all lower urinary tract activity. Similar effects were seenfor conantokin T.

Further effects of Con G and Con T on bladder contraction amplitude andon EUS EM G activity are shown in FIGS. 17 and 18. The conantokinsappear to be more discriminatory in their inhibitory effect on striatedsphincter than on bladder, compared to other NMDA antagonists. Thus, itis possible to dose the conantokins in such a manner to selectivelydecrease bladder/ sphincter dyssynergia in spinal cord-injured patients.

Example 19 Biological Activity of Conantokin Peptide Derivatives andConantokin Peptide Chimeras

Several conantokin peptide derivatives and conantokin peptide chimeraswere prepared using conventional techniques and their inhibitoryactivity was measured using the spermine-stimulated [³H]MK-801 bindingassay as described by Zhou et al. (1996). The results are shown in Table17. A value for IC₅₀ of <100 μM has been found to have activity in thein vivo assays described herein.

TABLE 17 Inhibitory Activity of Con G Derivatives and Chimeras TestSubstance Sequence IC₅₀ (μM) Con G G E γ γ L Q γ N Q γ L I R γ K S N0.195 A³ A 9.1 Y³ Y 0.96 S³ S 3.6 S_(p) ³ S_(p) 0.9 A⁴ A ≧20 S⁴ S ≧20S_(p) ⁴ S_(p) ≧20 E⁴ Y ≧20 A⁷ A 0.045 Y⁷ Y 0.96 S⁷ S 0.25 S_(p) ⁷ S_(p)1.2 A¹⁰ A 1.1 S¹⁰ S 1.8 S_(p) ¹⁰ S_(p) 0.56 A¹⁴ A 0.16 S¹⁴ S 0.16 S_(p)¹⁴ S_(p) 1.59 A⁷,Y¹⁰ A Y 0.079 A^(7,10,14) A A A 0.088 E^(7,10,14) E E E≧20 A^(2,7,10,14) A A A A ND A^(3,7,10,14) A A A A ND A^(4,7,10,14) A AA A ND Y³,S⁴,A^(7,10,14) Y S A A A ND Con G G E γ γ L Q γ N Q γ L I R γK S N 0.195 (1-10) Δ Δ Δ Δ Δ Δ Δ ND (A^(7,10,14))5-17 Δ Δ Δ Δ A A A ND5-17 Δ Δ Δ Δ ND (A^(10,14))6-17 Δ Δ Δ Δ Δ A A ND (A¹⁴)12-17 Δ Δ Δ Δ Δ ΔΔ Δ Δ Δ Δ A ND 12-17 Δ Δ Δ Δ Δ Δ Δ Δ Δ Δ Δ ND (A^(7,10,14))1-16 A A A Δ0.037 (A^(7,10,14))1-15 A A A Δ Δ 0.034 (A^(7,10,14))1-14 A A A Δ Δ Δ0.232 (A^(7,10))1-13 A A Δ Δ Δ Δ 0.253 (A^(7,10))1-12 A A Δ Δ Δ Δ Δ 0.30(A^(7,10,14))2-17 Δ A A A inactive (A^(7,10,14))3-17 Δ Δ A A A inactive(A^(7,10,14))4-17 Δ Δ Δ A A A inactive (A^(7,10,14))5-17 Δ Δ Δ Δ A A Ainactive (A^(7,10,14))6-17 Δ Δ Δ Δ Δ A A A inactive Con G(T)₂ Y Q K M LND Con G(L)₄ D A V N ND Con G(T)₂₋₃ Y Q K M L N L ND Con G(R)₂₋₃ V A K MAA A ND All derivatives have an amide C-terminal. γ is γ-carboxyglutamicacid. S_(p) is phosphoserine. Δ indicates a deletion of residue. ND isnot determined.

Example 20 Biological Stability of Conantokins

The stability of Con G, Con T, Con R and several synthetic derivativeswas determined in different biological media comprising serum (FS, 20%)and homogenates of liver (FL, 5%), kidney (FK, 25%) and brain (FB, 25%)from Frings audiogenic mice. The stability of Con G in normal saline aspH ≈6 was also examined The biological fluid was diluted to appropriatepercentage with RPMI-1640 cell culture media and preincubated for 15 minat 37° C. in water bath. Peptides (1 mg/ml stock) were added to thebiological media to a concentration of 50 μg/ml and incubated at 37° C.Peptide aliquots (100 μl) were removed at timed intervals and added to200 μl of ice cold 6% trifluoroacetic acid (TFA) and chilled on ice atleast 15 min. Supernatant was recovered following centrifugation at14,000×g for 3 min and frozen until analysis. Two hundred μl sample wasinjected into a HPLC system comprising 2 Dynamax Model SD-300 solventdelivery pumps, Rheodyne injection port, 5 ml injection loop, mixer andbubble suppression unit (Dynamax), Vydac T18TPS4 protein and peptide C₁₈column with precolumn and prefilter, Dynamax absorbance detector ModelUV-D11 and Dell Pentium computer with Dynamax chromatography software.The mobile phase used 0.1% TFA/H₂O as Buffer A and 0.1% TFA/acetonitrileas Buffer B. The following gradient was utilized:

Time (min) Flow Rate (ml/min) % Buffer A % Buffer B 0 1 95 5 3 1 95 5 251 45 55 27 1 45 55 29 1 5 95 33 1 5 95

The peak height and retention time was measured with Dynamax software.The results are shown in FIGS 19 and 20. Con G, [Ala⁷]Con G, Con-T andCon R were stable in all biological media for up to 4 hours. Thederivatives ECon G and ECon R (Gla residues replaced by Glu residuesdemonstrated rapid degradation in FL (T_(½)=25.5 sec and 2.4 min,respectively). The derivative ECon G demonstrated rapid degradation inFB (T_(½)=1.6 min). The results for the stability of Con G in normalsaline is shown in FIG. 21. Approximately 60% of the Con G is left after14 days.

Example 21 In vivo Activity of Conantokins on Preliminary Screening

The conantokins were originally described as producing a sleep-likestate in mice younger than two weeks old (Haack et al., 1990). Theconantokins are screened for biological activity using a modified formof this test. Several measures are assessed to measure the degree of thesleep-like state. Catalepsy, sleepy and righting reflex tests aredesigned to quantify the “sleep-like” behavior noted by Rivier et al.(1987). In all three assays, young (<2 weeks old) mice are given afree-hand i.c.v. dose (100 pmol/g in 10 μl) of a compound. Behaviors areassessed at 30 and 60 minutes post-injection.

Catalepsy Test: Young mice are positioned such that the two front pawsare placed on an overturned Petri dish. A mouse is considered catalepticif it fails to remove its paws in a 30 second period. Sleepy Test: Youngmice are observed without interference. If no activity is noted, theanimal is nudged with a gloved finger. A mouse is considered “sleepy” ifit makes no attempt to move away from the finger. Righting Reflex: Youngmice are positioned on their backs with legs in the air. A mouse isconsidered to have lost its “righting reflex” if it fails to rightitself (return to its normal position with paws on the floor) in 10seconds, thus “3/4” means three out of four mice lost righting reflex.In general, the ability of relatively high doses of conantokins toinduce a sleep-like state is correlated with affinity and efficacy inthe spermine-stimulated [³H]MK-801 binding assay described in Example19. The results are shown in Table 18.

TABLE 18 Conantokin Catalepsy Test^(a) Sleepy Test^(b) RightingReflex^(c) dose¹ 30 min 60 min 30 min 60 min 30 min 60 min Con G 4/4 2/44/4 4/4 2/4 2/4 Con T 3/4 1/4 4/4 4/4 2/4 2/4 Con R 2/3 2/3 2/3 2/3 2/32/3 Con L 4/4 3/4 4/4 4/4 2/4 3/4 Con Oc 0/3 0/3 2/3 0/3 0/3 0/3 Saline0/2 0/2 0/2 0/2 0/2 0/2 ¹˜2 week old mice, 5-7 g, sex not checked,i.c.v. dose of 100 pmol/g (600 pmol in 12 μl) or 12 μl of normal 0.9%)saline ^(a)positive if mice leave forepaws on Petri dish for >30 s^(b)positive if mice do not respond by moving away or sniffing after afinger poke ^(c)positive if mice fail to right themselves in <10 s

Example 22 In vivo Activity of Conantokins in Pain Models

The anti-pain activity of conantokin is shown in several animal models.These models include the nerve injury model (Chaplan, et al., 1997), thenocioceptive response to s.c. formalin injection in rats (Codene, 1993)and an NMDA-induced persistent pain model (Liu, et al., 1997). In eachof these models it is seen that the conantokin peptides, conantokinpeptide derivatives and conantokin peptide chimeras have analgesicproperties.

More specifically, this study evaluates the effect of intrathecaladministration of conantokins in mice models of nocioceptive andneuropathic pain. For nocioceptive pain, the effect of the conantokinsis studied in two different tests of inflammatory pain. The first is theformalin test, ideal because it produces a relatively short-lived, butreliable pain behavior that is readily quantified. There are two phasesof pain behavior, the second of which is presumed to result largely fromformalin-evoked inflammation of the hind paw. Conantokins areadministered 10 minutes prior to injection of formalin. The number offlinches and/or the duration of licking produced by the injection ismonitored. Since the first phase is presumed to be due to directactivation of primary afferents, and thus less dependent on long termchanges in the spinal cord, the conantokins are presumed to havegreatest effect on the magnitude of pain behavior in the second phase.

The mechanical and thermal thresholds in animals that received aninjection of complete Freund's adjuvant into the hind paw are alsostudied. This produces a localized inflammation including swelling ofthe hind paw and a profound decrease in mechanical and thermalthresholds, that are detected within 24 hours after injection. Thechanges in thresholds in rats that receive the conantokins are comparedwith those of rats that receive vehicle intrathecal injections.

To evaluate the contribution of long term, NMDA receptor-mediatedchanges to neuropathic (i.e., nerve injury-induced) behavior, amodification of the Seltzer model of pain that has been adapted for themouse is used. A partial transection of the sciatic nerve is first made.This also produces a significant drop in mechanical and thermalthresholds of the partially denervated hind paw. In general, themechanical changes are more profound. They peak around 3 days aftersurgery and persist for months.

An important issue is whether the drugs are effective when administeredafter the pain model has been established, or whether they are effectiveonly if used as a pretreatment. Clearly, the clinical need is for drugsthat are effective after the pain has developed. To address this issue,animals are studied in which the conantokin is administered repeatedly,after the inflammation (CFA) or nerve injury has been established. Inthese experiments, the conantokins are injected daily by the intrathecal(i.t.) route. The mechanical and thermal thresholds (measured,respectively, with von Frey hairs in freely moving animals and with theHargreave's test, also in freely moving animals) are repeated for a 2 to4 week period after the injury is induced and the changes in painmeasured monitored over time.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

LIST OF REFERENCES

Abiko, H. et al. (1986). Protective effect of phenytoin and enhancementof its action by combined administration of mannitol and vitamin E incerebral ischemia. Brain Res. 38:328-335.

Aldrete, J. A. et al. (1 979). Effect of pretreatment with thiopentaland phenytoin on postischemic brain damage in rabbits. Crit. Care Med.7:466-470.

Ascher, P. and Nowak, L. (1986). Calcium permeation of the channelsactivated by N-methyl-D-aspartate (NMDA) in mouse central neurons. J.Physiol. 377:35p.

Bach, F. W. (1994). Studies on the spinal pharmacology of a new model ofallydonia. Ann. Neurol. 36:288A.

Bergman, H. et al. (1990). Reversal of experimental Parkinsonism bylesions of the subthalamic nucleus. Science 249:1436-1438.

Bilsky E. J., et al. (1996). Competitive and non-competitive NMDAantagonists block the development of antinociceptive tolerance tomorphine, but not to selective mu or delta opioid agonists in mice. Pain68:229-237.

Bliss, et al. (1993). Nature 361:31.

Bodansky et al. (1966). Chem. Ind 38:1597-98.

Bormann, J. (1989). Memantine is a potent blocker ofN-methyl-D-aspartate (NMDA) receptor channels. Euro. J. Pharmacol.166:591-592.

Bowyer, J. F. (1982). Phencyclidine inhibition of the rate of kindlingdevelopment. Esp. Neurol. 75:173-175.

Chandler, P. et al. (1993). Polyamine-like Actions of Peptides Derivedfrom Conantokin-G, an N-methyl-D-aspartate (NMDA) Antagonist. J. Biol.Chem. 268:17173-17178.

Chaplan S. R. (1997). Efficacy of spinal NMDA receptor antagonism informalin hyperalgesia and nerve injury evoked allodynia in the rat. JPharmacol. Exp. Ther. 280:829-838.

Chapman, V. (1994). Bi-directional effects of intrathecal NMDA andsubstance P on rat dorsal dorn neuronal responses. Neurosci. Lett.178:90-94.

Cline, H. T. et al. (1987). N-Methyl-D-aspartate receptor antagonistdesegregates eye-specific stripes. Proc. Natl. Acad. Sci. USA84:4342-4345.

Codere, T. J. (1993). Eur. J. Neurosci. 5:390-393.

Codere, T. J. and Meizack, R. (1992). The contribution of excitatoryamnio acids to central sensitization and persistent nociception afterformalin-induced tissue injury. J Neurosci. 12:3665-3670.

Coderre, T. J. and Van Emple, I. (1992). The utility of excitatory aminoacid (EAA) antagonists as analgesic agents. I. Comparison of theantinociceptive activity of various classes of EAA antagonists inmechanical, thermal and chemical nonciceptive tests. Pain 59:345-352.

Collinridge, G. L. et al. (1983). Excitatory amino acids in synaptictransmission in the Schaffer collateral-commissural pathway of the rathippocampus. J. Physiol. 334:3446.

Cruz, L. J. et al. (1987). Conus geographus toxins that discriminatebetween neuronal and muscle sodium channels. J. Biol. Chem.260:9280-9288.

Davies, S. N. and Lodge, D. (1987). Evidence for involvement ofN-methylaspartate receptors in “wind up” of class 2 neurones in thedorsal horn of the rat. Brain Res.424:402-406.

Dickenson, A. H. and Sullivan, A. F. (1987). Evidence for a role of theNMDA receptor in the frequency dependent potentiation of deep rat dorsalhorn nociceptive neurones following C fibre stimulation.Neuropharmacology 26:1235-1238.

Dougherty, P. M. and Willia, W. D. (1991). Modification of the responsesof primate spinothalamic neurons to mechanical stimulation by excitatoryamino acids and an N-methyl-D-aspartate antagonist. Brain Res.542:15-22.

Doyle, D. D. et al. (1993). Divalent cation competition with[³H]saxitoxin binding to tetrodotoxin-resistant and -sensitive sodiumchannels. J. Gen. Physiol. 101:153-182.

Dudley, S. C. et al. (1995). A μ-Conotoxin-Insensitive Na⁺ ChannelMutant: Possible Localization of a Binding Site at the Outer Vestibule.Biophys. J. 69:1657-1665.

Dunbar, S. and Yaksh, T. L. (1996). Concurrent spinal infusion of MK801blocks spinal tolerance and dependence induced by chronic intrathecalmorphine in the rat. Anesthesiology 84:1177-1188.

Dunham, M. S. and Miya, T. A. (1957). J. Am. Pharm. Ass. Sci. Ed.46:208.

Elliott, K. et al. (1994). The NMDA receptor antagonists, LY274614 andMK-801, and the nitric oxide synthase inhibitor, NG-nitro-L-arginine,attenuate analgesic tolerance to the mu-opioid morphine but not to kappaopioids. Pain 56:69-75.

Evoniuk et al. (1991). Psychopharmacology 105:125-128.

Finney, D. J. (1971). Probit Analysis, Cambridge University Press,London.

Gray, W. R. (1993). Disulfide Structures of Highly Bridged Peptides: ANew Strategy for Analysis. Protein Science 2:1732-1748.

Greenamyre, J. T. and O'Brien, C. F. (1991). N-methyl-D-aspartateantagonists in the treatment of Parkinson's disease. Arch. Neurol.48:977-981.

Gutstein, H. B. and Trujillo, K. A. (1993). MK-801 inhibits thedevelopment of morphine tolerance at spinal sites. Brain Res.626:332-334.

Haack, J. A. et al. (1990). Conantokin-T: a gamma-carboxyglutamatecontaining peptide with N-methyl-d-aspartate antagonist activity. J.Biol. Chem. 265:6025-6029.

Harris, E. W. et al. (1984). Long-term potentiation in the hippocampusinvolves activation of N-methyl-D-aspartate receptors. Brain Res.323:132-137.

Heyes, M. P., et al. (1989). Cerebrospinal fluid quinolinic acidconcentrations are increased in acquired immune deficiency syndrome.Ann. Neurol. 26: 275-277.

Horiki, K. et al. (1978). Chemistry Letters 165-68.

Johnson, J. W. and Ascher, P. (1987). Glycine potentiates the NMDAresponse in cultured mouse brain neurons. Nature 325:529-531.

Johnson et al. (1990). Ann. Rev. Pharmacol. Toxicol. 30:707-750.

Kaiser et al. (1970). Anal. Biochem. 34:595.

Kapoor (1970). J. Pharm. Sci. 59:1-27.

Kest, B. et al. (1993). The NMDA receptor antagonist MK-801 protectsagainst the development of morphine tolerance after intrathecaladministration. Proc. West Pharmacol. Soc. 36:307-310.

Kleckner, N. W. and Dingledine, R. D. (1988). Requirement for glycineinactivation of NMDA receptors expressed in Xenopus oocytes. Science241:835-837.

Klockgether, T. et al. (1990). NMDS antagonists potentiateantiparkinsonian actions of L-dopa in monoamine-depleted rats. Ann.Neurol. 28:539-546.

Kornreich, W. D. et al. (1986). U.S. Pat. No. 4,569,967.

Lipton, S. A. (1996). Similarity of neuronal cell injury and death inAIDS dementia and focal cerebral ischemia: potential treatment with NMDAopen-channel blockers and nitric oxide-related species. Brain Pathol6:507-517.

Lipton, S. A. (1994). Neuronal injury associated with HIV-1 andpotential treatment with calcium-channel and NMDA antagonists. DavNeurosci. 61:145-151.

Liu, H. et al. (1997). NMDA-receptor regulation of substance P releasefrom primary afferent nociceptors. Nature 386:721-724.

Lutfy, K. et al. (1995). Blockade of morphine tolerance by ACEA-1328, anovel NMDA receptor/glycine site antagonist. Eur. J. Pharmacol.273:187-189.

Lutfy, K. et al. (1996). Inhibition of morphine tolerance by NMDAreceptor antagonists in the formalin test. Brain Res. 731:171-181.

Malmberg, A. B. and Taksh, T. L. (1995). The effect of morphine onformalin-evoked behavior and spinal release of excitatory amino acidsand prostaglandin E2 using microdialysis in conscious rats. Br. J.Pharmacol. 114:1069-1075.

Malmberg, A. B. and Taksh, T. L. (1992). Hyperalgesia mediated by spinalglutamate or substance-P receptor blocked by spinal cyclooxygenaseinhibition. Science 257:1276-1279.

Mao, J. et al. (1995). Experimental mononeuropathy reduces theantinociceptive effects of morphine: implications for commonintracellular mechanisms involved in morphine tolerance and neuropathicpain. Pain 61:353-364.

Mao, J. et al. (1994). Thermal hyperalgesia in association with thedevelopment of morphine tolerance in rats: roles of excitatory aminoacid receptors and protein kinase C. J Neurosci. 14:2301-2312.

Mayer, M. L. et al. (1987). Agonist- and voltage-gated calcium entry incultured mouse spinal cord neurons under voltage clamp measured usingarsenazo III. J. Neurosci. 7:3230-3244.

Mena, E. E. et al. (1990). Conantokin-G: a novel peptide antagonist tothe N-methyl-D-aspartic acid (NMDA) receptor. Neurosci. Lett.118:241-244.

McNamara, J. O. et al. (1988). Anticonvulsant and antiepileptogenicaction of MK-801 in the kindling and electroshock models.Neuropharmacology 27:563-568.

The Merck Manual of Diagnosis and Therapy, 16 Ed., Berkow, R. et al.,eds., Merck Research Laboratories, Rahway, N.J., pp. 1436-1445 (1992).

Methoden der Organischen Chemie (Houben-Weyl): Synthese von Peptiden, E.Wunsch (Ed.), Georg Thieme Verlag, Stuttgart, Ger. (1974).

Millian, M. J. and Seguin, L. (1994). Chemically-diverse ligands at theglycine B site coupled to N-methyl-D-asparatate (NMDA) receptorsselectively block the late phase of formalin-induced pain in mice.Neurosci. Lett. 178:139-143.

Morris, R. G. M. et al. (1986). Selective impairment and blockade oflong-term potentiation by an N-methyl-D-aspartate receptor antagonist,AP5. Nature 319:774-776.

Muller, W. E. et al. (1996). Neurotoxicity in rat cortical cells causedby N-methyl-D-aspartate (NMDA) and gp 120 of HIV-1: induction andpharmacological intervention. Prog Mol Subcell Biol. 16:44-57.

Nehlig, A. et al. (1990). Effects of phenobarbital in the developing ratbrain. In Neonatal Seizures, Wasterlain, C. G. and Vertt, P. (eds.),Raven Press, New York, pp. 285-194.

Neugebauer, V. et al. (1993). N-methyl-D-aspartate (NMDA) and non-NMDAreceptor antagonist block the hyperexcitability of dorsal horn neuronsduring development of acute arthritis in rat's kneejoint. JNeurophysiol. 70:1365-1377.

Nishida, K. et al. (1996). Increased brain levels of platelet-activatingfactor in a murine acquired immune deficiency syndrome are NMDAreceptor-mediated. J Neurochem 66:433-435.

Nishiuchi, Y. et al. (1993). Synthesis of gamma-carboxyglutanicacid-containing peptides by the Boc strategy. Int. J. Pept. Protein Res.42:533-538.

Nowak, L. et al. (1984). Magnesium gates glutamic-activated channels inmouse central neurons. Nature 307:462-465.

Olney, J. W. et al. (9187). Antiparkinsonian agents are phencyclidineagonists and N-methyl-D-aspartate antagonists. Eur. J. Pharmacol.142:319-320.

Olivera, B. M. et al. (1984). U.S. Pat. No. 4,447,356.

Olivera, B. M. et al. (1985). Peptide neurotoxins from fish-hunting conesnails. Science 230:1338-1343.

Park, C. K. et al. (1988). The glutamate antagonist MK-801 reduces focalischemia brain damage in the rat. Ann. Neurol. 24:543-551.

Popik, P. et al. (1995). 100 years of ibogaine: neurochemical andpharmacological actions of a putative anti-additive drug. Pharmacol.Rev. 47:235-253.

Raber, J. et al. (1996). Central nervous system expression of HIV-1 Gp120 activates the hypothalamic-pituitary-adrenal axis: evidence forinvolvement of NMDA receptors and nitric oxide synthase. Virology226:362-373.

Rall T. W. and Schleifer, L. S. in Goodman and Gilman's ThePharmacological Basis of Therapeutics, Seventh Ed., Gilman, A. G. etal., eds., Macmillan Publishing Co., New York, pp. 446-472 (1985).

Reynolds, I. J. et al. (1987). ³H-Labeled MK-801 binding to excitatoryamino acid receptor complex from rat brain is enhance by glycine. Proc.Natl. Acad. Sci. USA 84:7744-7748.

Rice, A. S. and McMahon, S. B. (1994). Pre-emptive intrathecaladministration of an NMDA receptor antagonist (AP-5) preventshyper-reflexia in a model of persistent visceral pain. Pain 57:335-340.

Rivier, J. R. et al. (1978). Biopolymers 17:1927-38.

Rivier, J. R. et al. (1987). Biochemistry, 26:8508-8512.

Rivier, J. R. et al. (1987). Total synthesis and furthercharacterization of the gamma-carboxyglutamate-containing ‘sleeper’peptide from Conus geographus. Biochem. 26:8508-8512.

Roberts et al. (1983). The Peptides 5:342-429.

Rytik, P. G. et al. (1991). Susceptibility of primary human glialfibrillary acidic protein-positive brain cells to human immunodeficiencyvirus infection in vitro. Anti-HIV activity of memantine. AIDS Res HumRetrovir 7:89-95.

Sambrook, J. et al. (1979). Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

Schroder & Lubke (1965). The Peptides 1:72-75, Academic Press, NY.

Sei, Y. et al. (1996). Quinolinic acid levels in an murineretrovirus-induced immunodeficiency syndrome. J Neurochem. 66:296-302.

Shimoyama, N. et al. (1996). Ketamine attenuates and reverses morphinetolerance in rodents. Anesthesiology 85:1357-1366.

Simon, R. P. et al. (1984). Blockade of N-methyl-D-aspartate receptorsmay protect against ischemic damage in the brain. Science 226:850-852.

Skolnick, P. et al. (1992). Noncompetitive Inhibition ofN-Methyl-D-Aspartate by Conantokin-G: Evidence for an AllostericInteraction at Polyamines Sites. J. Neurochem. 59:1526-1521.

Sluka, K. A. and Westland, K. N. (1992). An experimental arthritis inrats: dorsal horn aspartate and glutamate increases. Neurosci. Lett.145:141-144.

Spanagel, R. and Zieglgansberger, W. (1997). Anti-craving compounds forethanol: new pharmacological tools to study addictive processes. TrendsPharmacol. Sci. 18:54-59.

Standaert, D. C. et al. (1994). Organization of N-methyl-D-aspartateglutamate receptor gene expression in the basal ganglia of the rat. J.Comp. Neurol. 343:1-16.

Sweetman, P. M. (1993). The envelope glycoprotein of HIV-1 alters NMDAreceptor function. Eur. J. Neurosci. 5:276-283.

Starr, M. S. (1995). Antiparkinsonian actions of glutamateantagonists—alone and with L-Dopa: A review of evidence and suggestionsfor possible mechanisms. J. Neural Tans.[P-D Sect] 10:141-185.

Stewart and Young, Solid-Phase Peptide Synthesis, Freeman & Co., SanFrancisco, Calif. (1969).

Thompson, S. W. N. et al. (1990). Activity-dependent changes in ratventral horn neurones in vitro; summation of prolonged afferent evokedpostsynaptic depolarizations produce a d-APV sensitive windup. Eur. JNeurosci. 2:638-649.

Tiseo, P. J. et al. (1994). Modulation of morphine tolerance by thecompetitive N-methyl-D-aspartate receptor antagonist LY274614:assessment of opioid receptor changes. J. Pharmacol. Exp. Ther.268:195-201.

Tiseo, P. J. and Inturrisi, C. E. (1993). Attenuation and reversal ofmorphine tolerance by the competitive N-methyl-D-Aspartate receptorantagonist, LY274614. J Pharmacol Exp. Ther. 264:1090-1096.

Troupin, A. S. et al. (1986). MK-801. In New Anticonvulsant Drugs,Current Problems in Epilepsy 4, Meldrum, B. S. and Porter, R. J. (eds.),John Libbey, London, pp. 191-202.

Trujillo, K. A. and Akil, H. (1994). Inhibition of opiate tolerance bynon-competitive N-methyl-D-aspartate receptor antagonists. Brain Res.633:178-188.

Trujillo, K. A. and Akil, H. Excitatory acids and drugs of abuse: a rolefor N-methyl-D-aspartate receptors in drug tolerance, sensitization andphysical dependence. Drug Alcohol Depend. 38:139-154.

Ungerstedt, U. et al. (1973). Animal Models of Parkinsonism. In Advancesin Neurology: Progress in the Treatment of Parkinsonism, Calne, D. B.,Ed., Raven Press, New York, pp 257-271.

Vale et al. (1978). U.S. Pat. No. 4,105,603.

White, H. S., et al. (1992). Anticonvulsant profile of MDL 27,266: anorally active, broad-spectrum anticonvulsant agent. Epilepsy Res.12:217-226.

White, H. S., et al. (1995). Experimental Selection, Quantification, andEvaluation of Antiepileptic Drugs. In Antiepileptic Drugs, 4th Ed.,Levy, R. H., eds., Raven Press, N.Y., pp. 99-110.

Williams, K. et al. (1991). Modulation of the NMDA receptor bypolyamines (Minireview). Life Sci. 48:469-498.

Wong, C. S. et al. (1996). Effects of NMDA receptor antagonists oninhibition of morphine tolerance in rats: binding at mu-opioidreceptors. Eur. J Pharmacol 297:27-33.

Wong, E. H. P. et al. (1986). The anticonvulsant MK-801 is a potent NMDAantagonist. Proc. Natl. Acad. Sci. USA 83:7104-7108.

Wroblewski, J. T. et al. (1989). Glycine and D-serine act a positivemodulators of signal transduction at N-methyl-D-aspartate sensitiveglutamate receptors in cultured cerebellar granule cells.Neuropharmacology 28:447452.

Yamamoto, T. and Yaksh, T. L. (1992). Comparison of the antinociceptiveeffects of pre- and posttreatment with intrathecal morphine and MK-801,an NMDA antagonist, on the formalin test in the rat. Anesthesiology77:757-763.

Zhou L. M., et al. (1996). Synthetic Analogues of Conantokin-G: NMDAAntagonists Acting Through a Novel Polyamine-Coupled Site. J. Neurochem.66:620-628.

Zigmond, M. J. et al. (1987). Parkinsonism: Insights from animal modelsutilizing neurotoxic agents. In Animal Models of Demential, Coyle, J.T., Ed., Alan R. Liss, Inc., pp 1-38.

U.S. Pat. No. 3,972,859 (1976).

U.S. Pat. No. 3,842,067 (1974).

U.S. Pat. No. 3,862,925 (1975).

U.S. Pat. No. 5,550,050 (1996).

Published PCT Application WO 92/19195 (1992).

Published PCT Application WO 94/25503 (1994).

Published PCT Application WO 95/01203 (1995).

Published PCT Application WO 95/05452 (1995).

Published PCT Application WO 96/02286 (1996).

Published PCT Application WO 96/02646 (1996).

Published PCT Application WO 96/40871 (1996).

Published PCT Application WO 96/40959 (1996).

Published PCT Application WO 97/12635 (1997).

71 17 amino acids amino acid <Unknown> linear peptide Conus geographusModified-site 3..14 /note= “Xaa at residues 3 and 4 isgamma-carboxyglutamic acid; Xaa at residues 7, 10 and 14 may be anyamino acid but is preferably gamma-carboxyglutamic acid” 1 Gly Glu XaaXaa Leu Gln Xaa Asn Gln Xaa Leu Ile Arg Xaa Lys Ser 1 5 10 15 Asn 21amino acids amino acid <Unknown> linear peptide Conus tulipaModified-site 3..13 /note= “Xaa at residues 3 and 4 isgamma-carboxyglutamic acid; Xaa at reisdues 10 and 14 may be any aminoacid but is preferably gamma-carboxyglutamic acid” 2 Gly Glu Xaa Xaa TyrGln Lys Met Leu Xaa Asn Leu Arg Xaa Ala Glu 1 5 10 15 Val Lys Lys AsnAla 20 19 amino acids amino acid <Unknown> linear peptide Conus lynceusModified-site 3..15 /note= “Xaa at residues 3 and 4 isgamma-carboxyglutamic acid; Xaa at residues 11 and 15 may be any aminoacid, but is preferably gamma-carboxyglutamic acid” 3 Gly Glu Xaa XaaVal Ala Lys Met Ala Ala Xaa Leu Ala Arg Xaa Asp 1 5 10 15 Ala Val Asn 27amino acids amino acid <Unknown> linear peptide Conus radiatusModified-site 3..15 /note= “Xaa at residues 3 and 4 isgamma-carboxyglutamic acid; Xaa at residues 11 and 15 may be any aminoacid, but is preferably gamma-carboxyglutamic acid” Disulfide-bond21..25 4 Gly Glu Xaa Xaa Val Ala Lys Met Ala Ala Xaa Leu Ala Arg Xaa Asn1 5 10 15 Ile Ala Lys Gly Cys Lys Val Asn Cys Tyr Pro 20 25 24 aminoacids amino acid <Unknown> linear peptide Conus sulcatus Modified-site3..14 /note= “Xaa at residues 3 and 4 is gamma-carboxyglutamic acid; Xaaat residues 10 and 14 may be any amino acid, but is preferablygamma-carboxyglutamic acid” 5 Gly Asp Xaa Xaa Tyr Ser Lys Phe Ile XaaArg Glu Arg Xaa Ala Gly 1 5 10 15 Arg Leu Asp Leu Ser Lys Phe Pro 20 23amino acids amino acid <Unknown> linear peptide Conus orchroleucusModified-site 3..19 /note= “Xaa at residues 3 and 4 isgamma-carboxyglutamic acid; Xaa at residues 11 and 19 may be any aminoacid, but is preferably gamma-carboxyglutamic acid” 6 Gly Glu Xaa XaaTyr Arg Lys Ala Met Ala Xaa Leu Glu Ala Lys Lys 1 5 10 15 Ala Gln XaaAla Leu Lys Ala 20 19 amino acids amino acid <Unknown> linear peptideConus gloriamaris Modified-site 3..14 /note= “Xaa at residue 4 isgamma-carboxyglutamic acid; Xaa at residues 10 and 14 may be any aminoacid, but is preferably gamma-carboxy glutamic acid” 7 Gly Ala Lys XaaAsp Arg Asn Asn Ala Xaa Ala Val Arg Xaa Arg Leu 1 5 10 15 Glu Glu Ile 18amino acids amino acid <Unknown> linear peptide Conus caracteristicusModified-site 3..14 /note= “Xaa at residues 3 and 4 isgamma-carboxyglutamic acid; Xaa at residues 7, 10 and 14 may be anyamino acid, but is preferably gamma-carboxyglutamic acid” 8 Gly Tyr XaaXaa Asp Arg Xaa Ile Ala Xaa Thr Val Arg Xaa Leu Glu 1 5 10 15 Glu Ala 18amino acids amino acid <Unknown> linear peptide Conus quercinusModified-site 3..14 /note= “Xaa at residues 3 and 4 isgamma-carboxyglutamic acid; Xaa at residues 7, 10 and 14 may be anyamino acid, but is preferably gamma-carboxyglutamic acid” 9 Gly Tyr XaaXaa Asp Arg Xaa Val Ala Xaa Thr Val Arg Xaa Leu Asp 1 5 10 15 Ala Ala 5amino acids amino acid <Unknown> linear peptide internal 10 Lys Pro GlyArg Lys 1 5 6 amino acids amino acid <Unknown> linear peptide internal11 Lys Pro Gly Arg Lys Asn 1 5 4 amino acids amino acid <Unknown> linearpeptide N-terminal Modified-site 3..4 /note= “Xaa isgamma-carboxyglutamic acid” 12 Gly Glu Xaa Xaa 1 6 amino acids aminoacid <Unknown> linear peptide internal Modified-site 3..6 /note= “Xaa isgamma-carboxyglutamic acid” 13 Leu Gln Xaa Asn Gln Xaa 1 5 4 amino acidsamino acid <Unknown> linear peptide internal Modified-site /note= “Xaais gamma-carboxyglutamic acid” 14 Leu Ile Arg Xaa 1 6 amino acids aminoacid <Unknown> linear peptide internal Modified-site /note= “Xaa isgamma-carboxyglutamic acid” 15 Tyr Gln Lys Met Leu Xaa 1 5 4 amino acidsamino acid <Unknown> linear peptide internal Modified-site /note= “Xaais gamma-carboxyglutamic acid” 16 Asn Leu Arg Xaa 1 7 amino acids aminoacid <Unknown> linear peptide C-terminal 17 Ala Glu Val Lys Lys Asn Ala1 5 7 amino acids amino acid <Unknown> linear peptide internalModified-site /note= “Xaa is gamma-carboxyglutamic acid” 18 Val Ala LysMet Ala Ala Xaa 1 5 4 amino acids amino acid <Unknown> linear peptideinternal Modified-site /note= “Xaa is gamma-carboxyglutamic acid” 19 LeuAla Arg Xaa 1 12 amino acids amino acid <Unknown> linear peptideC-terminal 20 Asn Ile Ala Lys Gly Cys Lys Val Asn Cys Tyr Pro 1 5 10 4amino acids amino acid <Unknown> linear peptide C-terminal 21 Asp AlaVal Asn 1 6 amino acids amino acid <Unknown> linear peptide internal 22Leu Gln Ala Asn Gln Ala 1 5 4 amino acids amino acid <Unknown> linearpeptide internal 23 Leu Ile Arg Ala 1 6 amino acids amino acid <Unknown>linear peptide internal Modified-site /note= “Xaa isgamma-carboxyglutamic acid” 24 Leu Gln Ala Asn Gln Xaa 1 5 6 amino acidsamino acid <Unknown> linear peptide internal Modified-site /note= “Xaais gamma-carboxyglutamic acid” 25 Leu Gln Ser Asn Gln Xaa 1 5 6 aminoacids amino acid <Unknown> linear peptide internal Modified-site /note=“Xaa is gamma-carboxyglutamic acid” 26 Leu Gln Thr Asn Gln Xaa 1 5 4amino acids amino acid <Unknown> linear peptide N-terminal Modified-site3..4 /note= “Xaa is gamma-carboxyglutamic acid” 27 Gly Asp Xaa Xaa 1 6amino acids amino acid <Unknown> linear peptide internal Modified-site/note= “Xaa is gamma-carboxyglutamic acid” 28 Tyr Ser Lys Phe Ile Xaa 15 4 amino acids amino acid <Unknown> linear peptide internalModified-site /note= “Xaa is gamma-carboxyglutamic acid” 29 Arg Glu ArgXaa 1 10 amino acids amino acid <Unknown> linear peptide C-terminal 30Ala Gly Arg Leu Asp Leu Ser Lys Phe Pro 1 5 10 7 amino acids amino acid<Unknown> linear peptide internal Modified-site /note= “Xaa isgamma-carboxyglutamic acid” 31 Tyr Arg Lys Ala Met Ala Xaa 1 5 8 aminoacids amino acid <Unknown> linear peptide internal Modified-site /note=“Xaa is gamma-carboxyglutamic acid” 32 Leu Glu Ala Lys Lys Ala Gln Xaa 15 4 amino acids amino acid <Unknown> linear peptide C-terminal 33 AlaLeu Lys Ala 1 4 amino acids amino acid <Unknown> linear peptideN-terminal Modified-site 3..4 /note= “Xaa is gamma-carboxyglutamic acid”34 Gly Tyr Xaa Xaa 1 6 amino acids amino acid <Unknown> linear peptideinternal Modified-site 3..6 /note= “Xaa is gamma-carboxyglutamic acid”35 Asp Arg Xaa Val Ala Xaa 1 5 4 amino acids amino acid <Unknown> linearpeptide internal Modified-site /note= “Xaa is gamma-carboxyglutamicacid” 36 Thr Val Arg Xaa 1 4 amino acids amino acid <Unknown> linearpeptide C-terminal 37 Leu Asp Ala Ala 1 6 amino acids amino acid<Unknown> linear peptide internal Modified-site 3..6 /note= “Xaa isgamma-carboxyglutamic acid” 38 Asp Arg Xaa Ile Ala Xaa 1 5 4 amino acidsamino acid <Unknown> linear peptide C-terminal 39 Leu Glu Glu Ala 1 4amino acids amino acid <Unknown> linear peptide N-terminal Modified-site/note= “Xaa is gamma-carboxyglutamic acid” 40 Gly Ala Lys Xaa 1 6 aminoacids amino acid <Unknown> linear peptide internal Modified-site /note=“Xaa is gamma-carboxyglutamic acid” 41 Asp Arg Asn Asn Ala Xaa 1 5 4amino acids amino acid <Unknown> linear peptide internal Modified-site/note= “Xaa is gamma-carboxyglutamic acid” 42 Ala Val Arg Xaa 1 5 aminoacids amino acid <Unknown> linear peptide C-terminal 43 Arg Leu Glu GluIle 1 5 17 base pairs nucleic acid single linear other nucleic acid/desc = “probe” 44 CARGARAAYC ARGARYT 17 718 base pairs nucleic acidsingle linear DNA (cDNA) Conus geographus CDS 110..409 45 GCGCCTTGCCTGAGGAACGA CGTGTCTTCC CCTGCCCTCT CTGTCTTCCT GACTGCAGCC 60 TTGAGCCACCCAGCCGTCAT CTCTACCATC GACTTCACCC TGATTGGCG ATG CAC 115 Met His 1 CTG TACACG TAT CTG TAT CTG CTG GTG CCC CTG GTG ACC TTC CAC CTA 163 Leu Tyr ThrTyr Leu Tyr Leu Leu Val Pro Leu Val Thr Phe His Leu 5 10 15 ATC CTA GGCACG GGC ACA CTA GAT GAT GGA GGC GCA CTG ACT GAA CGC 211 Ile Leu Gly ThrGly Thr Leu Asp Asp Gly Gly Ala Leu Thr Glu Arg 20 25 30 CGT TCA GCT GACGCC ACA GCG CTG AAA GCT GAG CCT GTC CTC CTG CAG 259 Arg Ser Ala Asp AlaThr Ala Leu Lys Ala Glu Pro Val Leu Leu Gln 35 40 45 50 AAA TCC GCT GCCCGC AGC ACC GAC GAC AAT GGC AAG GAC AGG TTG ACT 307 Lys Ser Ala Ala ArgSer Thr Asp Asp Asn Gly Lys Asp Arg Leu Thr 55 60 65 CAG ATG AAG AGG ATTCTC AAA CAG CGA GGA AAC AAA GCC AGA GGC GAA 355 Gln Met Lys Arg Ile LeuLys Gln Arg Gly Asn Lys Ala Arg Gly Glu 70 75 80 GAA GAA GTT CAA GAG AATCAG GAA TTG ATC AGA GAA AAA AGT AAT GGA 403 Glu Glu Val Gln Glu Asn GlnGlu Leu Ile Arg Glu Lys Ser Asn Gly 85 90 95 AAA AGA TAATCAAGCTGGTGTTCCAC GTTATACCCG TCAGTTCTAA AATCCCCAGA 459 Lys Arg 100 TAGATCGTTCCCTATTTTTG CCACATTCTT TCTTTCTCTT TTCATTTAAT TCCCCAAATA 519 TTTCATGTTTATTCTCACGT AATTGTAAAA TTTTTAGGAG GAATGGTGTG TGTGTATGTG 579 CAAACTGTATCATACATAAA TAATGCGAAT TTAAGGAAGA AATTTTGCAG ATCCATGCAC 639 AGAAAGTCGTTAAAGACAAA TTGTATGAAT AACCAAATTT GATTTGAATC AATAAAGAAC 699 CCACTAAGTGAAAAAAAAA 718 100 amino acids amino acid linear protein 46 Met His LeuTyr Thr Tyr Leu Tyr Leu Leu Val Pro Leu Val Thr Phe 1 5 10 15 His LeuIle Leu Gly Thr Gly Thr Leu Asp Asp Gly Gly Ala Leu Thr 20 25 30 Glu ArgArg Ser Ala Asp Ala Thr Ala Leu Lys Ala Glu Pro Val Leu 35 40 45 Leu GlnLys Ser Ala Ala Arg Ser Thr Asp Asp Asn Gly Lys Asp Arg 50 55 60 Leu ThrGln Met Lys Arg Ile Leu Lys Gln Arg Gly Asn Lys Ala Arg 65 70 75 80 GlyGlu Glu Glu Val Gln Glu Asn Gln Glu Leu Ile Arg Glu Lys Ser 85 90 95 AsnGly Lys Arg 100 17 base pairs nucleic acid single linear other nucleicacid /desc = “probe” 47 CCYTTNGCDA TRTTYTC 17 16 base pairs nucleic acidsingle linear other nucleic acid /desc = “probe” 48 GCCGTGCCTA GGATTA 16580 base pairs nucleic acid single linear DNA (cDNA) Conus radiatus CDS127..447 49 TTCTGTCAGT TCAGATTTCG CCGTGCCCGA GGAACGACGT GTCTTCCCTTGCTCTCTCCA 60 TCTTCCTGAC AGCAGCTTTG AGCCACCCAG CCGTCATCTC TGCCGTCGACTTCACCCTGA 120 TTGGCG ATG CAA CTG TAC ACG TAT CTG TAT CTG CTG GTG TCCCTG GTG 168 Met Gln Leu Tyr Thr Tyr Leu Tyr Leu Leu Val Ser Leu Val 1 510 ACC TTC TAC CTA ATC CTA GGC ACG GGC ACG CTA GGT CAT GGA GGC GCA 216Thr Phe Tyr Leu Ile Leu Gly Thr Gly Thr Leu Gly His Gly Gly Ala 15 20 2530 CTG ACT GAA CGC CGT TCG ACT GAC GCC ACA GCA CTG AAA CCT GAA CCT 264Leu Thr Glu Arg Arg Ser Thr Asp Ala Thr Ala Leu Lys Pro Glu Pro 35 40 45GTC CTC CTG CAG AAA TCC TCT GCC CGC AGC ACC GAC GAC AAT GGC AAC 312 ValLeu Leu Gln Lys Ser Ser Ala Arg Ser Thr Asp Asp Asn Gly Asn 50 55 60 GACAGG TTG ACT CAG ATG AAG AGG ATT CTC AAA AAG CGA GGA AAC AAA 360 Asp ArgLeu Thr Gln Met Lys Arg Ile Leu Lys Lys Arg Gly Asn Lys 65 70 75 GCC AGAGGA GAA GAA GAA GTT GCA AAA ATG GCG GCA GAG CTT GCC AGA 408 Ala Arg GlyGlu Glu Glu Val Ala Lys Met Ala Ala Glu Leu Ala Arg 80 85 90 GAA AAC ATTGCA AAA GGC TGT AAA GTT AAT TGT TAC CCG TGACACTCGT 457 Glu Asn Ile AlaLys Gly Cys Lys Val Asn Cys Tyr Pro 95 100 105 CAGTTCTAAA GTCCCCAGATAGATCGTTCC CTATTTTTGC CACATTCTTT CTTTCTCTTT 517 TCATTTAATT CCCCAAATCTTTCATGTCTA TTCTCACGTA AAGAATTTAA TTGTAGAATT 577 TTT 580 107 amino acidsamino acid linear protein 50 Met Gln Leu Tyr Thr Tyr Leu Tyr Leu Leu ValSer Leu Val Thr Phe 1 5 10 15 Tyr Leu Ile Leu Gly Thr Gly Thr Leu GlyHis Gly Gly Ala Leu Thr 20 25 30 Glu Arg Arg Ser Thr Asp Ala Thr Ala LeuLys Pro Glu Pro Val Leu 35 40 45 Leu Gln Lys Ser Ser Ala Arg Ser Thr AspAsp Asn Gly Asn Asp Arg 50 55 60 Leu Thr Gln Met Lys Arg Ile Leu Lys LysArg Gly Asn Lys Ala Arg 65 70 75 80 Gly Glu Glu Glu Val Ala Lys Met AlaAla Glu Leu Ala Arg Glu Asn 85 90 95 Ile Ala Lys Gly Cys Lys Val Asn CysTyr Pro 100 105 30 base pairs nucleic acid single linear other nucleicacid /desc = “primer” 51 TGCTCGAATA AACATGAAAG ATTTGGGGAA 30 27 basepairs nucleic acid single linear other nucleic acid /desc = “primer” 52TCTGCGATGC AACTGTACAC GTATCTG 27 394 base pairs nucleic acid singlelinear DNA (cDNA) Conus ochroleucus CDS 1..294 53 TAT CTG CTG GTG CCCCTG GTG ACC TTC CTC CTA ATC CTA GGC ACG GGC 48 Tyr Leu Leu Val Pro LeuVal Thr Phe Leu Leu Ile Leu Gly Thr Gly 1 5 10 15 ACA CTA GAT CAT GGAGGC GCA CTG ACT GAA CGC CGT TCG ACT GAC GCC 96 Thr Leu Asp His Gly GlyAla Leu Thr Glu Arg Arg Ser Thr Asp Ala 20 25 30 ATA GCA CTG AAA CCT GAGCCT GTC CTC CTG CAG AAA TCC TCT GCC CGC 144 Ile Ala Leu Lys Pro Glu ProVal Leu Leu Gln Lys Ser Ser Ala Arg 35 40 45 AGC ACC GAC GAC AAT GGC GGCGAC AGG TTG ACT CAG ATG AAG AGG ATT 192 Ser Thr Asp Asp Asn Gly Gly AspArg Leu Thr Gln Met Lys Arg Ile 50 55 60 CTC AAA AAG CGA GGA AAC AAA GCCAGA GGC GAA GAA GAA TAT AGA AAA 240 Leu Lys Lys Arg Gly Asn Lys Ala ArgGly Glu Glu Glu Tyr Arg Lys 65 70 75 80 GCG ATG GCA GAG CTC GAA GCT AAAAAA GCT CAA GAA GCT CTA AAG GCG 288 Ala Met Ala Glu Leu Glu Ala Lys LysAla Gln Glu Ala Leu Lys Ala 85 90 95 GGA CGA TAATCAAGTT GGGTGTTCCACGTGACACTC GTCAGTTCTA AAGTCCCCAG 344 Gly Arg ATAGATCGTT CCCTATTTTTGCCACATTCT TTCTTTCTCT TTTCATTTAA 394 98 amino acids amino acid linearprotein 54 Tyr Leu Leu Val Pro Leu Val Thr Phe Leu Leu Ile Leu Gly ThrGly 1 5 10 15 Thr Leu Asp His Gly Gly Ala Leu Thr Glu Arg Arg Ser ThrAsp Ala 20 25 30 Ile Ala Leu Lys Pro Glu Pro Val Leu Leu Gln Lys Ser SerAla Arg 35 40 45 Ser Thr Asp Asp Asn Gly Gly Asp Arg Leu Thr Gln Met LysArg Ile 50 55 60 Leu Lys Lys Arg Gly Asn Lys Ala Arg Gly Glu Glu Glu TyrArg Lys 65 70 75 80 Ala Met Ala Glu Leu Glu Ala Lys Lys Ala Gln Glu AlaLeu Lys Ala 85 90 95 Gly Arg 472 base pairs nucleic acid single linearDNA (cDNA) Conus sulcatus CDS 6..314 55 GGGCG ATG CAA CTG TAC ACG TATCTG TAT CTG CTG GTG CCC CTG GTG 47 Met Gln Leu Tyr Thr Tyr Leu Tyr LeuLeu Val Pro Leu Val 1 5 10 ACC TTC CAC CTA ATC CTA GGC ACG GGC ACA CTAGAT CAT GGA GGC GCA 95 Thr Phe His Leu Ile Leu Gly Thr Gly Thr Leu AspHis Gly Gly Ala 15 20 25 30 CTG ACT GAA CGC CGT TCG ACT GAC GCC ACA GCACTG AAA CCT GAG CCT 143 Leu Thr Glu Arg Arg Ser Thr Asp Ala Thr Ala LeuLys Pro Glu Pro 35 40 45 GTC CTG CAG AAA TCC GCT GCC CGC AGC ACC GAC GACAAT GGC AAG GAC 191 Val Leu Gln Lys Ser Ala Ala Arg Ser Thr Asp Asp AsnGly Lys Asp 50 55 60 AGG CTG ACT CAG ATG AAG AGG ATT CTC AAA AAG CGA GGAAAG AAT GCC 239 Arg Leu Thr Gln Met Lys Arg Ile Leu Lys Lys Arg Gly LysAsn Ala 65 70 75 CGT GGC GAT GAA GAA TAT TCA AAG TTT ATA GAG AGA GAA CGCGAA GCA 287 Arg Gly Asp Glu Glu Tyr Ser Lys Phe Ile Glu Arg Glu Arg GluAla 80 85 90 GGA AGA CTG GAT TTG TCA AAA TTC CCG TGACACTCGT CAGTTCTAAA334 Gly Arg Leu Asp Leu Ser Lys Phe Pro 95 100 ATCCCCAGAT AGATCGTTCCCTATTTTTGT CACATTCTTT CTTTCTTTTT TCATTAATTC 394 CCCAAATCTT TCATGTTTATTCTCACGTAA TGAATTTAAT TGTAGAATTT TTAGGGGGAA 454 GGGGGGGGGG CGAAACTG 472103 amino acids amino acid linear protein 56 Met Gln Leu Tyr Thr Tyr LeuTyr Leu Leu Val Pro Leu Val Thr Phe 1 5 10 15 His Leu Ile Leu Gly ThrGly Thr Leu Asp His Gly Gly Ala Leu Thr 20 25 30 Glu Arg Arg Ser Thr AspAla Thr Ala Leu Lys Pro Glu Pro Val Leu 35 40 45 Gln Lys Ser Ala Ala ArgSer Thr Asp Asp Asn Gly Lys Asp Arg Leu 50 55 60 Thr Gln Met Lys Arg IleLeu Lys Lys Arg Gly Lys Asn Ala Arg Gly 65 70 75 80 Asp Glu Glu Tyr SerLys Phe Ile Glu Arg Glu Arg Glu Ala Gly Arg 85 90 95 Leu Asp Leu Ser LysPhe Pro 100 379 base pairs nucleic acid single linear DNA (cDNA) Conuslynceus CDS 1..282 57 TAT CTG CTG GTG CCC CTG GTG ACC TTC CAC CTA ATCCTA GGC ACG GGC 48 Tyr Leu Leu Val Pro Leu Val Thr Phe His Leu Ile LeuGly Thr Gly 1 5 10 15 ACA CTA GAT CAT GGA GGC GCA CTG ACT GAA CGC CGTTCG ACT GAC GCC 96 Thr Leu Asp His Gly Gly Ala Leu Thr Glu Arg Arg SerThr Asp Ala 20 25 30 ATA GCA CTG AAA CCT GAG CCT GTC CTC CTG CAG AAA TCCTCT GCC CGC 144 Ile Ala Leu Lys Pro Glu Pro Val Leu Leu Gln Lys Ser SerAla Arg 35 40 45 AGC ACC GAC GAC AAT GGC AAC GAC AGG TTG ACT CAG ATG AAGAGG ATT 192 Ser Thr Asp Asp Asn Gly Asn Asp Arg Leu Thr Gln Met Lys ArgIle 50 55 60 CTC AAA AAG CGA GGA AAC AAA GCC AGA GGC GAA GAG GAA GTT GCAAAA 240 Leu Lys Lys Arg Gly Asn Lys Ala Arg Gly Glu Glu Glu Val Ala Lys65 70 75 80 ATG GCG GCA GAG CTT GCC AGA GAA GAC GCT GTA AAT GGG AAA 282Met Ala Ala Glu Leu Ala Arg Glu Asp Ala Val Asn Gly Lys 85 90 TGATAATCAAGTTGGGTGTT CCACGTGACA CTCGTCAGTT CTAAAGTCCC CAGATAGATC 342 GTGCCCTATTTTTGCCACAT TCTTTCTTTC TCTTTTT 379 94 amino acids amino acid linearprotein 58 Tyr Leu Leu Val Pro Leu Val Thr Phe His Leu Ile Leu Gly ThrGly 1 5 10 15 Thr Leu Asp His Gly Gly Ala Leu Thr Glu Arg Arg Ser ThrAsp Ala 20 25 30 Ile Ala Leu Lys Pro Glu Pro Val Leu Leu Gln Lys Ser SerAla Arg 35 40 45 Ser Thr Asp Asp Asn Gly Asn Asp Arg Leu Thr Gln Met LysArg Ile 50 55 60 Leu Lys Lys Arg Gly Asn Lys Ala Arg Gly Glu Glu Glu ValAla Lys 65 70 75 80 Met Ala Ala Glu Leu Ala Arg Glu Asp Ala Val Asn GlyLys 85 90 31 base pairs nucleic acid single linear other nucleic acid/desc = “primer” 59 GGAATTCAAT AAACATGAAA GATTTGGGGA A 31 31 base pairsnucleic acid single linear other nucleic acid /desc = “primer” 60GGAATTCGCG ATGCAACTGT ACACGTATCT G 31 386 base pairs nucleic acid singlelinear DNA (cDNA) Conus gloriamaris CDS 1..285 61 TGT CTG CTG GTG CCCCTG GTG ACC CTC TAC GTA ATT CTA GGC ACG GGC 48 Cys Leu Leu Val Pro LeuVal Thr Leu Tyr Val Ile Leu Gly Thr Gly 1 5 10 15 ACA CTA GCT CAT GGAGGC GCA CTG ACC GAA CGC CGT TTG GCT CAC GCC 96 Thr Leu Ala His Gly GlyAla Leu Thr Glu Arg Arg Leu Ala His Ala 20 25 30 AGA GCA ATG GAA CCT GATCCT GTC CTC CTG CAG AAA TCC GCT GCC CGC 144 Arg Ala Met Glu Pro Asp ProVal Leu Leu Gln Lys Ser Ala Ala Arg 35 40 45 AGC ACC GAC GAC AAC GGC AAGGAC AGG ATG ACA CAG AGG AAG AGG ATT 192 Ser Thr Asp Asp Asn Gly Lys AspArg Met Thr Gln Arg Lys Arg Ile 50 55 60 CTC AAA AAG CGA GGA AAC ACG GCCAGA GGC GCG AAA GAA GAT AGA AAT 240 Leu Lys Lys Arg Gly Asn Thr Ala ArgGly Ala Lys Glu Asp Arg Asn 65 70 75 80 AAT GCG GAG GCT GTT AGA GAA AGACTC GAA GAA ATA GGA AAA AGA 285 Asn Ala Glu Ala Val Arg Glu Arg Leu GluGlu Ile Gly Lys Arg 85 90 95 TAATCAAGCT GGGTGTTTCA CGTGACACTC ATCAGTTCTAAAGTCCCCAG ATAGATCGTT 345 CCCTATTTTT GCCATATTTC TTTCTTTCTC TTTTCATTTA A386 95 amino acids amino acid linear protein 62 Cys Leu Leu Val Pro LeuVal Thr Leu Tyr Val Ile Leu Gly Thr Gly 1 5 10 15 Thr Leu Ala His GlyGly Ala Leu Thr Glu Arg Arg Leu Ala His Ala 20 25 30 Arg Ala Met Glu ProAsp Pro Val Leu Leu Gln Lys Ser Ala Ala Arg 35 40 45 Ser Thr Asp Asp AsnGly Lys Asp Arg Met Thr Gln Arg Lys Arg Ile 50 55 60 Leu Lys Lys Arg GlyAsn Thr Ala Arg Gly Ala Lys Glu Asp Arg Asn 65 70 75 80 Asn Ala Glu AlaVal Arg Glu Arg Leu Glu Glu Ile Gly Lys Arg 85 90 95 356 base pairsnucleic acid single linear DNA (cDNA) Conus caracteristicus CDS 1..27963 TAT CTG CTG GTG CCC CTG GTG GCC TTC CAC CTA ATC CTA GGC ACG GGC 48Tyr Leu Leu Val Pro Leu Val Ala Phe His Leu Ile Leu Gly Thr Gly 1 5 1015 ACG CTA GCT CAT GGA GAC GCA CTG ACT GAA CGC CGT TCG GCT GAT GCC 96Thr Leu Ala His Gly Asp Ala Leu Thr Glu Arg Arg Ser Ala Asp Ala 20 25 30ACA GCA CTG AAA CCT GAG CCT GTC CTC CTG CAG AAA TCC GCT GCC CGC 144 ThrAla Leu Lys Pro Glu Pro Val Leu Leu Gln Lys Ser Ala Ala Arg 35 40 45 AGCACT GAC GAC AAT GGC AAG GAC AGG TTG ACT CAG AGG AAG AGG ACT 192 Ser ThrAsp Asp Asn Gly Lys Asp Arg Leu Thr Gln Arg Lys Arg Thr 50 55 60 CTC AAAAAG CGA GGA AAC ATG GCC AGA GGC TAC GAA GAA GAT AGA GAG 240 Leu Lys LysArg Gly Asn Met Ala Arg Gly Tyr Glu Glu Asp Arg Glu 65 70 75 80 ATT GCGGAG ACT GTT AGA GAA CTC GAA GAA GCA GGA AAA TGAAAAAGAT 289 Ile Ala GluThr Val Arg Glu Leu Glu Glu Ala Gly Lys 85 90 AGTTCTAAAG TCCCCAGATATATCGTTCCC TATTTTTGCC ACATTCTTTC TTTCTCTTTT 349 ATTTTAA 356 93 aminoacids amino acid linear protein 64 Tyr Leu Leu Val Pro Leu Val Ala PheHis Leu Ile Leu Gly Thr Gly 1 5 10 15 Thr Leu Ala His Gly Asp Ala LeuThr Glu Arg Arg Ser Ala Asp Ala 20 25 30 Thr Ala Leu Lys Pro Glu Pro ValLeu Leu Gln Lys Ser Ala Ala Arg 35 40 45 Ser Thr Asp Asp Asn Gly Lys AspArg Leu Thr Gln Arg Lys Arg Thr 50 55 60 Leu Lys Lys Arg Gly Asn Met AlaArg Gly Tyr Glu Glu Asp Arg Glu 65 70 75 80 Ile Ala Glu Thr Val Arg GluLeu Glu Glu Ala Gly Lys 85 90 390 base pairs nucleic acid single linearDNA (cDNA) Conus quercinus CDS 1..285 65 TAT CTG CTG GTG CCC CTG GTG GCCTTC CAC CTA ATC CTA GGC ACG GGC 48 Tyr Leu Leu Val Pro Leu Val Ala PheHis Leu Ile Leu Gly Thr Gly 1 5 10 15 ACG CTA GCT CAT GGA GAC GCA CGGACT GAA CGC CGT TCG GCT GAC GCC 96 Thr Leu Ala His Gly Asp Ala Arg ThrGlu Arg Arg Ser Ala Asp Ala 20 25 30 ACA GCG CTG AAA CCT GAG CCT GTC CTCCTG CAG AAA TCC GCT GCC CGC 144 Thr Ala Leu Lys Pro Glu Pro Val Leu LeuGln Lys Ser Ala Ala Arg 35 40 45 AGC ACT GAC GAC AAT GAC AGG GAC AGG TTGACT CAG ATG AAG AGG ATT 192 Ser Thr Asp Asp Asn Asp Arg Asp Arg Leu ThrGln Met Lys Arg Ile 50 55 60 CTC AAA AAG CGA GGA AAC ACG GCC AGA GGC TACGAA GAA GAT AGA GAG 240 Leu Lys Lys Arg Gly Asn Thr Ala Arg Gly Tyr GluGlu Asp Arg Glu 65 70 75 80 GTT GCG GAG ACT GTC AGA GAA CTC GAC GCA GCAGGA AAA AGA AAA 285 Val Ala Glu Thr Val Arg Glu Leu Asp Ala Ala Gly LysArg Lys 85 90 95 TGATTAATCA AGCTGGGTGT TCCACTTGAC ACTCGTCAGT TCTAAAGTCACCAGATAGAT 345 CGTTCCCTGT TTTTGCCCGT TTTTTCTCTT TCACTTTTCA TTTAA 390 95amino acids amino acid linear protein 66 Tyr Leu Leu Val Pro Leu Val AlaPhe His Leu Ile Leu Gly Thr Gly 1 5 10 15 Thr Leu Ala His Gly Asp AlaArg Thr Glu Arg Arg Ser Ala Asp Ala 20 25 30 Thr Ala Leu Lys Pro Glu ProVal Leu Leu Gln Lys Ser Ala Ala Arg 35 40 45 Ser Thr Asp Asp Asn Asp ArgAsp Arg Leu Thr Gln Met Lys Arg Ile 50 55 60 Leu Lys Lys Arg Gly Asn ThrAla Arg Gly Tyr Glu Glu Asp Arg Glu 65 70 75 80 Val Ala Glu Thr Val ArgGlu Leu Asp Ala Ala Gly Lys Arg Lys 85 90 95 16 amino acids amino acid<Unknown> linear peptide N-terminal Modified-site /note= “Xaa is His orGln” Modified-site 12 /note= “Xaa is Pro or Ser” Modified-site 15 /note=“Xaa is Thr or Ala” Modified-site 16 /note= “Xaa is Leu or Phe” 67 MetXaa Leu Tyr Thr Tyr Leu Tyr Leu Leu Val Xaa Leu Val Xaa Xa 1 5 10 15 17amino acids amino acid <Unknown> linear peptide Conus caracteristicusModified-site 10..16 /note= “Xaa at residues 10, 12, 13 and 16 may beany amino acid, but is preferably gamma-carboxyglutamic acid” 68 Gly AsnAsp Val Asp Arg Lys Leu Ala Xaa Leu Xaa Xaa Leu Tyr Xa 1 5 10 15 Ile 4amino acids amino acid <Unknown> linear peptide N-terminal 69 Gly AsnAsp Val 1 6 amino acids amino acid <Unknown> linear peptide internalModified-site /note= “Xaa is gamma-carboxyglutamic acid” 70 Asp Arg LysLeu Ala Xaa 1 5 4 amino acids amino acid <Unknown> linear peptideC-terminal Modified-site /note= “Xaa is gamma-carboxyglutamic acid” 71Leu Tyr Xaa Ile 1

What is claimed is:
 1. A substantially pure conantokin comprising afirst, second, third and fourth domain, said first, second, third andfourth domains being contiguous, said first domain selected from thegroup consisting of GEγγ (SEQ ID NO:12), GDγγ (SEQ ID NO:27), GYγγ (SEQID NO:34), GAKγ (SEQ ID NO:40) and GNDV (SEQ ID NO:69), said seconddomain selected from the group consisting of LQγNQγ(SEQ ID NO:13),YQKMLγ (SEQ ID NO:15), VAKMAAγ (SEQ ID NO:18), LQANQA (SEQ ID NO:22),LQANQγ (SEQ ID NO:24), LQSNQγ (SEQ ID NO:25), LQTNQγ (SEQ ID NO:26),YSKFIγ (SEQ ID NO:28), YRKAMAγ (SEQ ID NO:31), DRγVAγ (SEQ ID NO: 35),DRγIAγ (SEQ ID NO:38), DRNNAγ (SEQ ID NO:41) and DRKLAγ (SEQ ID NO: 70),said third domain selected from the group consisting of NLRγ (SEQ IDNO:16), LARγ (SEQ ID NO:19), RERγ (SEQ ID NO:29), LEAKKAQγ (SEQ IDNO:32), TVRγ (SEQ ID NO:36), AVRγ (SEQ ID NO:42) and Lγγ, and saidfourth domain selected from the group consisting of KSN, DAVN (SEQ IDNO:21), AGRLDLSKFP (SEQ ID NO:30), ALKA (SEQ ID NO:33), LDAA (SEQ IDNO:37), LEEA (SEQ ID NO:39), RLEEI (SEQ ID NO(:43) and LYγI (SEQ IDNO:71), and wherein each of said γ residues in said second domain, saidthird domain and said fourth domain may independently be substituted byAla, Ser, Glu, Tyr or phospho-Ser (pSer).
 2. The substantially pureconantokin of claim 1 selected from the group consisting of conantokin Lhaving the amino acid sequenceGly-Glu-Xaa₁-Xaa₁-Val-Ala-Lys-Met-Ala-Ala-Xaa₂-Leu-Ala-Arg-Xaa₂-Asp-Ala-Val-Asn(SEQ ID NO:3), conantokin S1 having the amino acid sequenceGly-Asp-Xaa₁-Xaa₁-Tyr-Ser-Lys-Phe-Ile-Xaa₂-Arg-Glu-Arg-Xaa₂-Ala-Gly-Arg-Leu-Asp-Leu-Ser-Lys-Phe-Pro(SEQ ID NO:5), conantokin Oc having the amino acid sequence,Gly-Glu-Xaa₁-Xaa₁-Tyr-Arg-Lys-Ala-Met-Ala-Xaa₂-Leu-Glu-Ala-Lys-Lys-Ala-Gln-Xaa₂-Ala-Leu-Lys-Ala(SEQ ID NO:6), conantokin Gm having the amino acid sequenceGly-Ala-Lys-Xaa₁-Asp-Arg-Asn-Asn-Ala-Xaa₁-Ala-Val-Arg-Xaa₂-Arg-Leu-Glu-Glu-Ile(SEQ ID NO:7), conantokin Ca2 having the amino acid sequenceGly-Tyr-Xaa₁-Xaa₁-Asp-Arg-Xaa₂-Ile-Ala-Xaa₂-Thr-Val-Arg-Xaa₂-Leu-Glu-Glu-Ala(SEQ ID NO:8), conantokin Qu having the amino acid sequenceGly-Tyr-Xaa₁-Xaa₁-Asp-Arg-Xaa₂-Val-Ala-Xaa₂-Thr-Val-Arg-Xaa₂-Leu-Asp-Ala-Ala(SEQ ID NO:9), and conantokin Ca1 (Con Ca1) having the amino acidsequenceGly-Asn-Asp-Val-Asp-Arg-Lys-Leu-Ala-Xaa₂-Leu-Xaa₂-Xaa₂-Leu-Tyr-Xaa₂-Ile(SEQ ID NO:68), wherein Xaa₁ and Xaa₂ are γ-carboxyglutamic acid.
 3. Thesubstantially pure conantokin of claim 2, wherein said conantokin ismodified by deleting one to five of the C-terminal amino acid residues.4. The sustantially pure conantokin of claim 2 having the amino acidsequenceGly-Asp-Xaa₁-Xaa₁-Tyr-Ser-Lys-Phe-Ile-Xaa₂-Arg-Glu-Arg-Xaa₂-Ala-Gly-Arg-Leu-Asp-Leu-Ser-Lys-Phe-Pro(SEQ ID NO:5).
 5. The sustantially pure conantokin of claim 1 having theamino acid sequenceGly-Glu-Xaa₁-Xaa₁-Tyr-Arg-Lys-Ala-Met-Ala-Xaa₂-Leu-Glu-Ala-Lys-Lys-Ala-Gln-Xaa₂-Ala-Leu-Lys-Ala(SEQ ID NO:6).
 6. The sustantially pure conantokin of claim 1 having theamino acid sequenceGly-Glu-Xaa₁-Xaa₁-Val-Ala-Lys-Met-Ala-Ala-Xaa₂-Leu-Ala-Arg-Xaa₂-Asp-Ala-Val-Asn(SEQ ID NO:3).
 7. The sustantially pure conantokin of claim 2 having theamino acid sequenceGly-Ala-Lys-Xaa₁-Asp-Arg-Asn-Asn-Ala-Xaa₂-Ala-Val-Arg-Xaa₂-Arg-Leu-Glu-Glu-Ile(SEQ ID NO:7).
 8. The sustantially pure conantokin of claim 2 having theamino acid sequenceGly-Tyr-Xaa₁-Xaa₁-Asp-Arg-Xaa₂-Ile-Ala-Xaa₂-Thr-Val-Arg-Xaa₂-Leu-Glu-Glu-Ala(SEQ ID NO:8).
 9. The sustantially pure conantokin of claim 1 having theamino acid sequenceGly-Tyr-Xaa₁-Xaa₁-Asp-Arg-Xaa₂-Val-Ala-Xaa₂-Thr-Val-Arg-Xaa₂-Leu-Asp-Ala-Ala(SEQ ID NO:9).
 10. The substanially pure conantokin of claim 1 havingthe amino acid sequenceGly-Asn-Asp-Val-Asp-Arg-Lys-Leu-Ala-Xaa₂-Leu-Xaa₂-Xaa₂-Leu-Tyr-Xaa₂-Ile(SEQ ID NO:68).
 11. The substantially pure conantokin of claim 1selected from the group consisting of conantokin L having the amino acidsequenceGly-Glu-Xaa₁-Xaa₁-Val-Ala-Lys-Met-Ala-Ala-Xaa₂-Leu-Ala-Arg-Xaa₂-Asp-Ala-Val-Asn(SEQ ID NO:3), conantokin S1 having the amino acid sequenceGly-Asp-Xaa₁-Xaa₁-Tyr-Ser-Lys-Phe-Ile-Xaa₁-Arg-Glu-Arg-Xaa₂-Ala-Gly-Arg-Leu-Asp-Leu-Ser-Lys-Phe-Pro(SEQ ID NO:5), conantokin Oc having the amino acid sequenceGly-Glu-Xaa₁-Xaa₁-Tyr-Arg-Lys-Ala-Met-Ala-Xaa₂-Leu-Glu-Ala-Lys-Lys-Ala-Gln-Xaa₁-Ala-Leu-Lys-Ala(SEQ ID NO:6), conantokin Gm having the amino acid sequenceGly-Ala-Lys-Xaa₁-Asp-Arg-Asn-Asn-Ala-Xaa₂-Ala-Val-Arg-Xaa₂-Arg-Leu-Glu-Glu-Ile(SEQ ID NO:7), conantokin Ca2 having the amino acid sequenceGly-Tyr-Xaa₁-Xaa₁-Asp-Arg-Xaa₂-Ile-Ala-Xaa₂-Thr-Val-Arg-Xaa₂-Leu-Glu-Glu-Ala(SEQ ID NO:8), conantokin Qu having the amino acid sequenceGly-Tyr-Xaa₁-Xaa₁-Asp-Arg-Xaa₂-Val-Ala-Xaa₂-Thr-Val-Arg-Xaa₂-Leu-Asp-Ala-Ala(SEQ ID NO:9), and conantokin Ca1 (Con Ca1) having the amino acidsequenceGly-Asn-Asp-Val-Asp-Arg-Lys-Leu-Ala-Xaa₂-Leu-Xaa₂-Xaa₂-Leu-Tyr-Xaa₂-Ile(SEQ ID NO:68), wherein Xaa₁ is γ-carboxyglutamic acid and Xaa₂ isselected from the group consisting of Ser, Ala, Glu, Tyr and phospho-Ser(pSer).
 12. The substantially pure conantokin of claim 11, wherein saidconantokin is modified by deleting one to five of the C-terminal aminoacid residues.
 13. The substantially pure conantokin of claim 11,wherein said conantokin is modified by deleting one to five of theC-terminal amino acid residues.
 14. A substantially pure conantokinselected from the group consisting of conantokin T having the amino acidsequenceGly-Glu-Xaa₁-Xaa₁-Tyr-Gln-Lys-Met-Leu-Xaa₂-Asn-Leu-Arg-Xaa₂-Ala-Glu-Val-Lys-Lys-Asn-Ala(SEQ ID NO:2) and conantokin R having the amino acid sequenceGly-Glu-Xaa₁-Xaa₁-Val-Ala-Lys-Met-Ala-Ala-Xaa₂-Leu-Ala-Arg-Xaa₂-Asn-Ile-Ala-Lys-Gly-Cys-Lys-Val-Asn-Cys-Tyr-Pro(SEQ ID NO:4), wherein Xaa₁ is γ-carboxyglutamic acid and Xaa₂ isselected from the group consisting of Ser, Ala, Glu, Tyr and phospho-Ser(pSer).
 15. The substantially pure conantokin of claim 14, wherein saidconantokin is modified by deleting one to five of the C-terminal aminoacid residues.
 16. A substantially pure conantokin comprising a first,second, third and fourth domain, said first, second, third and fourthdomains being contiguous, said first domain selected from the groupconsisting of GEγγ (SEQ ID NO:12), GDγγ (SEQ ID NO:27), GYγγ (SEQ IDNO:34), GAKγ (SEQ ID NO:40) and GNDV (SEQ ID NO:69), said second domainselected from the group consisting of LQγNQγ (SEQ ID NO:13), YQKMLγ (SEQID NO:15), VAKMAAγ (SEQ ID NO:18), LQANQA (SEQ ID NO:22), LQANQγ (SEQ IDNO:24), LQSNQγ (SEQ ID NO:25), LQTNQγ (SEQ ID NO:26), YSKFIγ (SEQ IDNO:28), YRKAMAγ (SEQ ID NO:31), DRγVAγ (SEQ ID NO:35), DRγIAγ (SEQ IDNO:38), DRNNAγ (SEQ ID NO:41) and DRKLAγ (SEQ ID NO: 70), said thirddomain is LIRγ (SEQ ID NO:14), and said fourth domain selected from thegroup consisting of AEVKKNA (SEQ ID NO:17), NIAKGCKVNCYP (SEQ ID NO:20),DAVN (SEQ ID NO:21), AGRLDLSKFP (SEQ ID NO:30), ALKA (SEQ ID NO:33),LDAA (SEQ ID NO:37), LEEA (SEQ ID NO:39), RLEEI (SEQ ID NO:43) and LYγI(SEQ ID NO:71), and wherein each of said γ residues in said seconddomain, said third domain and said fourth domain may independently besubstituted by Ala, Ser, Glu, Tyr or phospho-Ser (pSer).
 17. Thesubstantially pure conantokin of claim 16, wherein said conantokin ismodified by deleting one to five of the C-terminal amino acid residues.